Antisense gene systems of pollination control for hybrid seed production

ABSTRACT

A process is described for producing hybrid seed using male-sterile plants created by employing molecular techniques to manipulate antisense DNA and other genes that are capable of controlling the production of fertile pollen in plants. Transformation techniques are used to introduce constructs containing antisense DNA and other genes into plants. Said plants are functionally male-sterile and are useful for the production of hybrid seed by the crossing of said male-sterile plants with pollen from male-fertile plants. Hybrid seed production is simplified and improved by this invention and can be extended to plant crop species for which commercially acceptable hybrid seed production methods are not currently available.

This application is a divisional of application Ser. No. 08/288,734,filed Aug. 12, 1994 which is a division of Ser. No. 07/892,635, filedJun. 2, 1992, now U.S. Pat. No. 5,356,799, which is a continuation ofSer. No. 07/306,438, filed Feb. 3, 1988, now abandoned, which is acontinuation-in-part if application Ser. No. 07/151,906, filed Feb. 3,1988 now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a method for producing male sterileplants and hybrid seed, to genetic material used to impart the malesterility trait and to new products produced by said method, namely,genetically transformed plants carrying the male sterile trait, malesterile plants and hybrid seed produced by pollinating said plants withpollen from male fertile plants.

Production of hybrid seed for commercial sale is a large industry.Hybrid plants grown from hybrid seed benefit from the heterotic effectsof crossing two genetically distinct breeding lines. The agronomicperformance of this offspring is superior to both parents, typically invigour, yield, and uniformity. The better performance of hybrid seedvarieties compared to open-pollinated varieties makes the hybrid seedmore attractive for farmers to plant and thereby commands a premiumprice in the market place.

In order to produce hybrid seed uncontaminated with selfed seedpollination control methods must be implemented to ensurecross-pollination and not self-pollination. Pollination controlmechanisms can be mechanical, chemical, or genetic.

A simple mechanical method for hybrid seed production can be used if theplant species in question has spatially separate male and female flowersor separate male and female plants. The corn plant, for example, haspollen producing male flowers in an inflorescence at the apex of theplant and female flowers in the axils of leaves along the stem.Outcrossing is assured by mechanically detasselling female plants toprevent selfing.

Most major crop plants of interest, however, have both functional maleand female organs within the same flower so emasculation is not a simpleprocedure. It is possible to remove by hand the pollen forming organsbefore pollen shed, however this form of hybrid seed production isextremely labour intensive and hence expensive. Seed is produced in thismanner if the value and amount of seed recovered warrants the effort.

A second general method of producing hybrid seed is to use chemicalsthat kill or block viable pollen formation. These chemicals, termedgametocides, are used to impart a transitory male-sterility. Commercialproduction of hybrid seed by use of gametocides is limited by theexpense and availability of the chemicals and the reliability and lengthof action of the applications. These chemicals are not effective forcrops with an extended flowering period because new flowers will beproduced that will not be affected. Repeated application of chemicals isimpractical because of costs.

Many current commercial hybrid seed production systems for field cropsrely on a genetic method of pollination control. Plants that are used asfemales either fail to make pollen, fail to shed pollen or producepollen that is biochemically unable to effect self-fertilization. Plantsthat are unable (by several different means) to self pollinatebiochemically are termed self-incompatible. Difficulties associated withthe use of self-incompatibilities are: availability and propagation ofthe self-incompatible female line and stability of theself-incompatibility. In some instances self-incompatibility can beovercome chemically or immature buds can be pollinated by hand beforethe biochemical mechanism that blocks pollen is activated.Self-incompatible systems that can be deactivated are often veryvulnerable to stressful climatic conditions that break or reduce theeffectiveness of the biochemical block to self-pollination.

Of more widespread interest for commercial seed production are systemsof pollen control based on genetic mechanisms causing male sterility.These systems are of two general types: (a) genic male sterility, whichis the failure of pollen formation because of one or more nuclear genesor (b) cytoplasmic-genetic male sterility (commonly called cytoplasmicmale sterility or CMS) in which pollen formation is blocked or abortedbecause of a defect in a cytoplasmic organelle (mitochondrion).

Nuclear (genic) sterility can be either dominant or recessive. Adominant sterility can only be used for hybrid seed formation ifpropagation of the female line is possible (for example, via in vitroclonal propogation). A recessive sterility could be used if sterile andfertile plants are easily discriminated. Commercial utility of genicsterility systems is limited however by the expense of clonalpropogation and roguing the female rows of self-fertile plants.

Many successful hybridization schemes involve the use of CMS. In thesesystems, a specific mutation in the cytoplasmically locatedmitochondrion can, when in the proper nuclear background, lead to thefailure of mature pollen formation. In some other instances, the nuclearbackground can compensate for the cytoplasmic mutation and normal pollenformation occurs. The nuclear trait that allows pollen formation inplants with CMS mitochondrial is called restoration and is the propertyof specific "restorer genes". Generally the use of CMS for commercialseed production involves the use of three breeding lines, themale-sterile line (female parent), a maintainer line which is isogenicto the male-sterile line but contains fully functional mitochondrial andthe male parent line.

The male parent line may carry the specific restorer genes (usuallydesignated a restorer line) which then imparts fertility to the hybridseed. For crops (eg. vegetables) for which seed recovery from the hybridis unimportant, a CMS system could be used without restoration. Forcrops for which the fruit or seed of the hybrid is the commercialproduct then the fertility of the hybrid seed must be restored byspecific restorer genes in the male parent or the male-sterile hybridmust be pollinated. Pollination of non-restored hybrids can be achievedby including with hybrids a small percentage of male fertile plants toeffect pollination. In most species, the CMS trait is inheritedmaternally (because all cytoplasmic organelles are inherited from theegg cell only), which can restrict the use of the system.

In a crop of particular interest herein, the oilseed crop of the speciesBrassica napus or Brassica campestris, commonly referred to as Canola,no commercial hybrid system has been perfected to date. Mechanicalemasculation of flowers is not practical for hybrid seed production onany scale. The use of currently available gametocides is impracticalbecause of the indeterminate nature of flower production. Repeatedapplication of chemicals is expensive and the method is prone tocontamination with selfed seed.

Genes that result in self-incompatibility are quite widespread inBrassica species and self-incompatible hybrid systems have been used forhybrid seed production in vegetables. Major difficulties are associatedwith the propagation of the female lines and the breakdown ofself-incompatibilities under stressful conditions. Adaptation of thesesystems to Brassica oilseeds is restricted by the expense of increasingthe female lines and the availability of appropriate self-incompatiblegenes in the dominant Canola species, Brassica napus.

A variety of sources of male sterility are available in Brassicaspecies. Both recessive and dominant genic systems have been reported,however their use is restricted because large scale in vitro propagationor roguing of female lines is in most cases impractical for large scaleseed production.

Additionally, a number of CMS systems have been reported in Brassicaspecies. Four of these systems have been explored as possible vehiclesfor hybrid seed production: pol, nap, anand and ogu. The Polima system(pol) has been widely studied and is probably the closest to commercialuse. Good restoration and maintenance of pol CMS has been achieved,however the system suffers from potential instability of the CMS withhigh temperature, a reduction in the hererotic effect of crossingdifferent lines (because of the defective mitochondria) and a reductionin hybrid seed oil content. The use of other CMS systems is alsorestricted by heat sensitivity (nap), difficulty in restoration offertility (ogu, anand), difficulty in the maintenance of the sterility(nap) and low temperature chlorosis associated with the sterilecytoplasm (ogu). Improvement of these systems is the object ofconsiderable research, however all of the systems have some inherentweaknesses that limit their utility.

An ideal system for hybrid seed production in any crop would be a formof genic male sterility that could be regulated to allow controlled malefertility for the propagation of the female line and geneticallyrestored by the male parent to allow the production of self-fertilehybrids.

SUMMARY OF INVENTION

It is an object of the invention as claimed herein, to provide a methodfor producing genic male sterility in plants. It is also an object ofthis invention to provide a method of producing genic male sterilitywhich can be readily adapted to the production of hybrid seed, includinghybrid seed with "restored" male fertility or male sterility which isnot expressed in the plants grown from said seed.

For certain crops of interest, such as vegetables, it is only the leavesstems or roots of the plant that are sold commercially. Therefore, eventhough the male sterility trait may be inherited and expressed in thehybrid plant it is not necessary to overcome or restore male fertilityin the seed of the hybrid plant. However, for other crops, the commodityof commerce is the seed or fruit produced by the hybrid plant. Thus foroptimal commercial utility of the hybrid it is desirable that the hybridis self-fertile.

In addition, the use of genic male sterile plants for commercial seedproduction requires the increase of seed of the genic male-sterile line(frequently called maintenance). Certain aspects of the invention asdescribed herein, relate to the increase or maintenance of seed withgenic male-sterility.

Specifically, it is an object of the present invention to provide amethod of producing hybrid seed in three stages as follows:

(a) producing a genetically transformed female parent by:

i) inserting into the genome of a plant cell of said pollen producingplant which is capable of being regenerated into a differentiated wholeplant, one or more recombinant DNA sequences comprising antisense DNAwhich block the production of functional pollen grains or render thedeveloping pollen grains susceptible to a chemical agent orphysiological stress which blocks the production of functional pollengrains.

ii) obtaining a transformed plant cell of said plant; and

iii) regenerating from said plant cell a plant which is geneticallytransformed with said DNA sequences described in (a)(i) above and whichis male sterile or carries the male sterile trait.

(b) increasing the number of female parent plants by:

i) fertilizing the genetically transformed plant described in step (a)with pollen produced by a suitable male fertile plant, in a manner toeffect a seed increase of genetically transformed plants; or

ii) clonal propagation of said genetically transformed plant describedin step (a) using tissue explants thereof, or other in vitro propagationtechniques;

(c) effecting a hybrid cross by pollinating said genetically transformedfemale parent plants with pollen produced by suitable male parent line.

According to one aspect of the present invention genic male sterilitymay be produced by transforming plant cells that are capable ofregeneration into a differentiated whole plant, with a recombinant DNAmolecule ("antisense DNA") that codes for RNA that is complimentary toand capable of hybridizing with the RNA encoded by a gene that iscritical to pollen formation or function (the "sense gene"), therebyinhibiting the expression of the sense gene. The antisense DNA ispreferably constructed by inverting the coding region of the sense generelative to its normal presentation for transcription to allow fortranscription of its complement, hence the complementariness of therespective RNAs encoded by these DNA's. In order to block the productionof mRNA produced by the sense gene, the antisense DNA should preferablybe expressed at approximately the same time as the sense gene. Thetiming must be approximate in the sense that the antisense RNA must bepresent within the cell to block the function of the RNA encoded by thesense gene. To accomplish this result, the coding region of theantisense DNA is preferably placed under the control of the samepromoter as found in the sense gene thereby causing both to betranscribed at the same time. The concept of regulating gene expressionusing antisense RNA is described in Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews--Trends in Genetics,Vol. 1(1) 1986.

Thus, according to one aspect of the invention as claimed herein, weprovide a method of producing a male sterile plant from a plant selectedfrom those species of pollen producing plants that are capable of beinggenetically transformed, which method comprises:

(a) identifying and isolating, preferably from said pollen producingplant, a sense gene or coding sequence that is critical to pollenformation or function in said plant;

(b) inserting into the genome of a plant cell of said plant that iscapable of being regenerated into a differentiated whole plant, arecombinant double stranded DNA molecule comprising:

(i) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by said sense gene or coding sequence;

(ii) a promoter which functions in said plant cell plant to causetranscription of said DNA sequence into RNA at about the time oftranscription of said sense gene or coding sequence; and preferably

(iii) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence.

(c) obtaining a transformed plant cell of said plant; and

(d) regenerating from said transformed plant cell a geneticallytransformed plant which is male sterile.

It is possible that the RNA transcribed from the antisense DNA will beeffective in blocking translation of the RNA encoded by the sense geneeven though a terminator sequence is not encoded by said recombinant DNAmolecule comprising the antisense DNA.

According to another aspect of the preceding method as claimed in theclaims, we provide a method of increasing the production of seed of thegenic male sterile line by transforming the plant of interest with anantisense gene that is linked to a gene that confers resistance to aselective agent. According to this scheme, it is possible to produce amale sterile line by crossing the genetically transformed plant (malesterile) with a suitable non-transformed male fertile plant and usingsaid selective agent to select for plants containing the antisense geneamong plants grown from seed produced from such a cross. Theoretically,any effective selective agent for which a resistance gene has beenidentified could be used within the scope of this aspect of theinvention, including but not limited to genes coding for resistance toherbicides and plant diseases. Such a selective agent could be said tofall within two broad non-mutually exclusive categories, a chemicalagent and a physiological stress. A chemical agent, such as a herbicide,could be used to produce male sterile plants on a commercial scale.Examples of herbicides for which a resistance gene has been identifiedare glyphosate (described in Comai, L., Facciotti, D., Hiatt, W. R.,Thompson, G., Rose, R. E., Stalker, D. M., 1985, Nature, Vol. 317, Pages741-744) and chlorsulfuron (described in Haughn, G. W., and Somerville,C. R., 1986, Mol. Gen. Genet., Vol. 210, Pages 430-434).

Thus, according to another aspect of the invention as claimed herein, weprovide a method of producing hybrid seed from plants selected fromthose species of pollen producing plants which are capable of beinggenetically transformed comprising the steps of:

(a) producing a genetically transformed plant which is made sterile by:

i) identifying and isolating, preferably from said pollen producingplant, a sense gene or a coding sequence that is critical to pollenformation or function in said plant;

ii) inserting into the genome of a plant cell of said plant that iscapable of regeneration into a differentiated whole plant, a gene whichconfers on said plant resistance to a chemical agent or physiologicalstress and, linked to said gene, a gene comprising:

(A) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by said sense gene or coding sequence;

(B) a promoter which functions in said plant cell to cause transcriptionof said DNA sequence into RNA at about the time of transcription of theRNA encoded by said sense gene or coding sequence; and preferably;

(C) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence;

iii) obtaining a transformed plant cell of the said plant; and

iv) regenerating from said transformed plant cell a plant which isgenetically transformed with the genes described in step (a) ii) above;and

(b) increasing the number of genetically transformed plants by:

i) crossing the genetically transformed plant described in step (a) iv)above with suitable male fertile plant;

ii) using the same chemical agent or physiological stress to eliminateplants which do not contain the genes described in step (a) ii) aboveamong plants grown from seed produced by such cross; and

iii) repeating such a cross over several generations with the plantsobtained as in step (b) ii) above in the presence of said chemical agentor physiological stress to increase the numbers of male sterile plants;

(c) effecting a hybrid cross by pollinating said male sterile plantswith pollen from suitable male fertile donors.

In a hybrid seed production scheme where there are alternating rows ofmale sterile plants and male fertile plants, it may be simpler but notessential to carry out the final selection of male steriles in the fieldalongside the male fertile donors. Therefore it is desirable if thesuitable male fertile donors are previously transformed to resistance tothe selective agent to avoid having to selectively apply said agent tothe rows of male sterile plants. Therefore step (c) would beaccomplished by growing seed produced from a cross between the selectedgenetically transformed plants and suitable male fertile donors,alongside seed of suitable male fertile donors which have previouslybeen made resistant to said chemical agent or physiological stress (theselective agent).

In accordance with the preceding methods, the invention is also directedto a plant containing a recombinant DNA molecule which comprises:

(a) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by a sense gene that is critical to pollen formation orfunction in said plant;

(b) a promoter which functions in said plant to cause transcription ofsaid DNA sequence into RNA that is at about the time of transcription ofthe RNA encoded by said sense gene; and preferably

(c) a terminator sequence that defines a termination signal duringtranscription of said DNA sequence.

In accordance with the preceding methods the invention is also directedto hybrid seed containing a recombinant DNA molecule which comprises:

(a) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by a sense gene that is critical to pollen formation orfunction in a plant grown from said seed;

(b) a promoter which functions in a plant grown from said seed to causetranscription of said DNA sequence into RNA at about the time oftranscription of the RNA encoded by said sense gene; and preferably

(c) a terminator sequence that defines a termination signal duringtranscription of said DNA sequence.

According to another aspect of the invention as claimed herein, weregulate the expression of the coding region of the antisense DNA byusing an inducible promoter. In this scheme, the promoter can be left inan induced state throughout pollen formation or at least for a periodwhich spans the period of transcription of the sense gene. A promoterthat is inducible by a simple chemical is particularly useful since themale sterile plant can easily be maintained by self-pollination whengrown in the absence of said chemical. Restoration is inherent ingrowing plants produced from hybrid seed in the absence of said inducer.

Thus according to another aspect of the invention as claimed herein weprovide a method of producing hybrid seed with restored fertility fromplants selected from those species of pollen producing plants which arecapable of being genetically transformed comprising the steps of:

(a) producing a genetically transformed plant which carries the malesterile trait by:

i) identifying and isolating, preferably from said pollen producingplant, a sense gene that is critical to pollen formation or function insaid plant;

ii) inserting into the genome of a plant cell of said plant that iscapable of regeneration into a differentiated whole plant, a recombinantdouble stranded DNA molecule comprising:

(A) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by said sense gene or coding sequence;

(B) an inducible promoter which can function in said plant cell to causetranscription of said DNA sequence into RNA during the time oftranscription of said sense gene or coding sequence; and preferably

(C) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence

iii) obtaining a transformed plant cell of said plant; and

iv) regenerating from said transformed plant cell a plant which isgenetically transformed with said double stranded DNA molecule describedin (ii) above and can be rendered male sterile by said inducer;

(b) increasing the number of genetically transformed plants by growingthe genetically transformed plant described in step (a)(iv) above in theabsence of the relevant inducer to produce a male-fertile plant,permitting self-fertilization and growing seed of such a plant, over anumber of generations, in the absence of the inducer to increase thenumber of genetically transformed plants;

(c) effecting a hybrid cross by growing said genetically transformedplants alongside plants of a suitable line of male fertile donors in thepresence of the relevant inducer during pollen formation in order toproduce male sterile plants and permit cross-pollination of said malesterile plants.

In accordance with step (a) of the preceding method we provide a methodto produce a plant which carries the male sterile trait.

In accordance with the preceding method, the invention is also directedto a plant which contains a recombinant DNA molecule comprising:

(a) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by a sense gene which is critical to pollen formationor function in said plant;

(b) an inducible promoter which functions in said plant to causetranscription of said DNA sequence into RNA during the time oftranscription of said gene; and

(c) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence.

In accordance with the preceding method the invention is also directedto hybrid seed containing a recombinant DNA molecule which comprises:

(a) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by a sense gene which is critical to pollen formationor function in a plant grown from said seed;

(b) an inducible promoter which functions in said plant to causetranscription of said DNA sequence into RNA during the time oftranscription of said sense gene; and

(c) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence. According to another aspect of theinvention as claimed herein, we provide a DNA coding sequence isolatedfrom a plant of the species Brassica napus cv. Westar which is expressedonly in microspores and whose expression is critical to microsporedevelopment. It is believed that this DNA fragment, the sequence ofwhich is shown in FIG. 2a, (nucleotides 600-2430) will be found andexpressed exclusively in pollen in other species of pollen-bearingplants, particularly species of plants within the genus Brassica and thefamily Cruciferae. The occurrence of this sequence in other species ofpollen-bearing plants may be routinely ascertained by knownhybridization techniques. It is believed that the similarity of plantgenes from species to species will allow for the preceding aspects ofthe present invention to be carried out using said DNA fragment in anynumber of pollen bearing plant species that are capable of beinggenetically transformed. The universality of plant genes has been widelydocumented in the literature and homologous plant genes have beendescribed for plant actins (Shah, D. M., et al, J. Mol. Appl. Genet.2:111-126, 1983), phytochrome (Hershey, H. P., et al., Proc. Natl. Acad.Sci. USA 81:2332-2337, 1984) storage proteins (Singh, N. K., et al.,Plant Mol. Biol. 11:633-639, 1988) enzymes such as glutamine synthase(Lightfoot, D. A., et al, Plant Mol. Biol. 11:191-202, 1988, andreferences within) and nitrate reductase (Cheng, C., et al, EMBO Jour.7:3309-3314). These and other examples in the literature clearlydemonstrate that many plant genes are highly conserved. It is also clearthat this conservation applies not only to structural proteins but toenzymatic proteins important to cellular physiology. Therefore, it isbelieved that said DNA fragment, when found in another plant species,will be critical to microspore development and will be able to beemployed to carry out the present invention in such species.Furthermore, it is to be understood that any number of different genesthat are crucial to microspore development or tissues uniquely involvedin and critical to microspore development could be isolated and used inaccordance with the preceding methods.

According to another aspect of the invention, we provide a pollenspecific promoter sequence (nucleotide 1-595 shown in FIG. 2a) that canbe utilized to limit the expression of any given DNA sequence to pollentissue and to a period during pollen formation. The pollen specificpromoter can be used to limit the expression of DNA sequences adjacentto it, to pollen tissue in both the Solanacae and Cruciferae families asprovided for in the specific examples and it is fully believed that thisDNA fragment or functional fragment thereof will function as a pollenspecific promoter in all pollen bearing plant species of interest thatare capable of being genetically transformed. This pollen specificpromoter can be used in conjunction with its naturally flanking codingsequence described above (nucleotides 600-2430) or any other codingsequence that is critical to pollen formation or function to carry outeach of the preceding aspects and embodiments of this invention which donot call for the use of an inducible promoter.

By using a pollen specific promoter to regulate the expression of theantisense DNA, it is possible to interfere with normal microsporedevelopment in any given plant, without having to first isolate from thegenomic DNA of said plant, a gene which codes for a developmentallyregulated protein that is critical to microspore development. We willdescribe a method to produce a male sterile plant where the sense genetargetted for inactivation is a gene that is critical to cellularfunction or development and is expressed in metabolically competent celltypes. To produce a male sterile plant, such a gene is specificallyinactivated in pollen by using a pollen specific promoter to limit thetranscription of its antisense DNA complement to pollen tissue. We willalso describe a method to produce a male-sterile plant, wherein thesense gene targeted for inactivation is a foreign gene which confers onthe plant resistance to a chemical agent or a naturally occurring orartificially induced physiological stress. This gene can be insertedinto the genome of the plant, prior to, after or concurrently with theantisense DNA. A male sterile plant can be produced by growing a plantin the presence of such a stress during pollen formation and using anantisense gene comprising said pollen specific promoter to specificallyinactivate, in pollen, during pollen formation, a gene conferring on theremainder of the plant resistance to said stress. Any stress which canadequately be controlled on a large scale and for which a resistancegene has been identified may theoretically be employed in this scheme,including possibly but not limited to herbicides, pathogenic organisms,certain antibiotics and toxic drugs. In this scheme, a male sterileplant line can be maintained by self-pollination when grown in theabsence of the biochemical or physiological stress. Restoration isinherent in growing plants produced from hybrid seed in the absence ofsaid stress.

It is expected that one may use any number of different pollen specificpromoters to carry out this invention. It is often difficult todetermine a priori what pollen specific promoter could be used toinhibit a gene that is critical to pollen development or cellularfunction and development, but certain conditions must be met. The pollenspecific promoter used to carry out the relevant aspects of thisinvention should be a promoter that functions to cause transcription ofthe antisense gene at a time concomitant with the expression of thesense gene sought to be inactivated. The pollen specific promoter shouldalso function as to produce sufficient levels of antisense RNA such thatthe levels of the sense gene product are reduced. Investigations of themechanism of antisense RNA inhibition of gene expression in modelsystems have suggested that equal or greater than equal levels ofantisense RNA may be required in order to observe a significantreduction of sense gene activity. However, in some cases it is notedthat low levels of antisense RNA can have a specific reduction in sensegene activity. Therefore it is suggested that the pollen specificpromoter that is used to carry out certain aspects of this invention bechosen based on the observation that the pollen specific promoterfunctions to cause the expression of any sequences adjacent to it to betranscribed at a time that parallels or overlaps the period of time thatthe sense gene sought to be inactivated is express and that the levelsof antisense RNA expressed from the antisense gene be of levelssufficient to inhibit the sense gene expression, usually to mean greaterthan or equal to the levels of sense RNA.

The determination of the most likely developmental stage in which thesense gene is targeted for inactivation can be accomplished by chosing atime in the developmental pattern of pollen formation at which the sensegene is maximally expressed and using a pollen specific promoter thatdisplays a similar developmental pattern.

Thus according to another aspect of our invention, we provide a methodof producing a male sterile plant from a plant selected from thosespecies of pollen bearing plants that are capable of being geneticallytransformed which method comprises the steps of:

(a) identifying and isolating, preferably from said pollen producingplant, a sense gene or coding sequence that is critical to cellularfunction or development and expressed in metabolically competent celltypes;

(b) inserting into the genome of a plant cell a recombinant doublestranded DNA molecule comprising:

i) a DNA sequence that codes for RNA that is complimentary to the RNAencoded by said sense gene or coding sequence;

ii) a pollen specific promoter which functions in said plant cell tocause transcription of said DNA sequence into RNA in a time frame whichenables said RNA to block the function of the RNA encoded by said sensegene or coding sequence; and preferably

iii) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence.

(c) obtaining a plant cell that has been genetically transformed withsaid DNA sequence;

(d) regenerating from said transformed plant cell a geneticallytransformed plant which is male sterile.

Similarly, in accordance with another aspect of this invention weprovide a method of producing hybrid seed from plants selected fromthose species of pollen producing plants which are capable of beinggenetically transformed comprising the steps of:

(a) producing a male sterile plant by:

i) identifying and isolating, preferably from said pollen producingplant, a sense gene or a coding sequence that is critical to cellularfunction or development and expressed in metabolically competent celltypes.

ii) inserting into the genome of a plant cell of said plant that iscapable of regeneration into a differentiated whole plant, a gene whichconfers on said plant resistance to a chemical agent or physiologicalstress and, linked to said gene, a recombinant DNA sequence comprising:

(A) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by said sense gene or coding sequence;

(B) a promoter which functions in said plant cell to cause transcriptionof said DNA sequence into RNA in a time frame which enables said RNA toblock the function of the RNA encoded by said sense gene or codingsequence; and preferably;

(C) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence

iii) obtaining a transformed plant cell of said plant; and

iv) regenerating from said transformed plant cell a plant which isgenetically transformed with the genes described in step (a)(ii) aboveand which is male sterile; and

(b) increasing the number of genetically transformed plants by:

i) crossing the genetically transformed plant described in step (a)(iv)above with a suitable male fertile plant;

ii) using a chemical agent or physiological stress to eliminate plantswhich do not contain the genes described in step (a)(ii) above amongplants grown from seed produced by such cross; and

iii) repeating such a cross over several generations with the plantsobtained as in step (b)(ii) above in the presence of said chemical agentor physiological stress to increase the numbers of male sterile plants;

(c) effecting a hybrid cross by pollinating said male sterile plantswith pollen from a suitable male fertile donors.

Again it is to be understood that where there are alternating rows ofmale sterile plants and male fertile plants, it may be simpler but notessential to carry out the final selection of male steriles in the fieldalongside the male fertile donors. Therefore it is desirable if thesuitable male fertile donors are previously transformed to be resistantto the selective agent to avoid having to selectively apply said agentto the rows of male sterile plants.

Therefore, in accordance with the two preceding aspects of thisinvention, the invention is also directed to a plant and hybrid seedcontaining DNA comprising a recombinant DNA molecule which comprises:

(a) a DNA sequence that codes for RNA that is complimentary to mRNAencoded by a gene that is essential to cellular function or developmentin metabolically competent cell types;

(b) a pollen specific promoter which functions in said plant or plantgrown from said hybrid seed to cause transcription of said DNA sequenceinto RNA in a time frame which enables said RNA to block the function ofthe RNA encoded by said gene; and preferably

(c) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence.

It is to be understood that a sense gene that codes for a protein thatis critical to cellular function or development may be identified in theliterature and isolated in a simplified fashion according to the methodsdescribed below.

Examples of such proteins are proteins such as actin, tublin orubiquitin, three proteins which are essential to cellular growth anddevelopment.

Sequences for actin genes isolated from plants have been published (forexample; Baird W. V., and Meagher, R. B., EMBO J. 6:3223-3231, 1987, orShah, D. M., Hightower, R. C. and Meagher, R. B., Proc Natl Acad Sci USA79:1022-1026, 1982) and actin is known to play a critical role in normalcellular function especially during mitosis and meiosis where actinforms part of the cellular apparatus for cellular division.

The sequence for plant tubulin has also been described (Raha, D., Sen,K. and Biswas, B. B. Plant Mol Biol 9:565-571, 1987). Tubulin, likeactin, is known to be important in the cellular life cycle particularlyin regards to cell shape, transport and spindle formation during mitosisand meiosis.

The DNA sequence for plant ubiquitin has also been published (Gausing,K. and Barkardottir, R. Eur J. Biochem 158:57-62, 1986). Ubiquitin is aprotein involved in the turnover of cellular proteins and as such has acritical role in the regulation of specific cellular protein levels. Inaddition, ubiquitin is one of the most highly conserved proteins ineukaryotic cells. Interference with ubiquitin expression can causeabnormalities in the turnover of cellular proteins.

If any of the aforementioned proteins are not present in the cell,proper cellular function is interfered with and the cell fails todevelop properly.

It is believed that a gene that is found to be essential to cellulargrowth or development in one plant species will have a similarcounterpart in other plant species, since it is generally understoodthat within the plant kingdom there are genes that are nearly identicalor very homologous involved in the basic processes that control or are aresult of cellular development. It is further believed that a gene thatinterferes with the expression of said gene (ie. an antisense gene) inone plant species will have the ability to do so in other plant species.

The similarity and universality of these genes have been exemplified inthe literature. The tissue-specific and developmentally regulatedexpression of a wheat endosperm protein synthesized in tobacco plantsgenetically transformed with this wheat gene has been reported (Flavell,R. B., et al, Second International Congress for Plant Molecular Biology,Abstract #97). In that example, the wheat gene functioned in the tobaccoplant in an identical fashion to the way in which it functions in awheat plant. Other literature clearly shows that the regulation of aspecific gene, which can be in many cases complex, is maintained intransgenic plants. One example of this is the phytochrome mediatedregulation of a wheat Chlorophyll a/b-binding protein in transgenictobacco (Nagy, F. et al, EMBO Jour. 5:1119-1124, 1986). In this examplethe light responsive specific regulation of the wheat gene wasmaintained in the foreign genetic environment. Not only do cereal genesfunction in a conserved manner, but genes from other plant species thatare more closely related maintain functionality in heterologous geneticsystems. Pea seed proteins are expressed properly in tobacco plants(Higgins, T. J. V., et al Plant Mol. Biol. 11:683-696, 1988), as aresoybean seed proteins, (Barker, S. J., et al, Proc. Natl. Acad. Sci. USA85:458-462, 1988) and pea rbcS genes (Nagy, F. et al., EMBO Jour.4:3063-3068, 1985). The scientific literature has numerous otherexamples of genes that have been used to genetically transform plantsand those genes maintain their ability to function properly in this newgenetic environment. Therefore the conserved nature of these genes, notonly in the DNA sequences which control the expression of these genes,but the actual protein structure coded for by these genes, is similaramong the plant species.

It follows that one should be able to specifically inhibit theproduction of these proteins by using antisense DNA which is specific tothe mRNAs encoded by the published sequences and a pollen specificpromoter according to the methods described above.

According to another aspect of the invention as claimed herein, weprovide a method of producing a male sterile plant from a plant selectedfrom those species of pollen producing plants which are capable of beinggenetically transformed, which method comprises the steps of:

(a) transforming a plant cell of said plant which is capable of beingregenerated into a differentiated whole plant with a sense gene whichconfers on said plant resistance to a chemical agent or a naturallyoccurring or artificially induced physiological or biochemical stress;

(b) regenerating from said transformed plant cell a geneticallytransformed plant which is resistant to the same stress;

(c) inserting into the genome of a plant cell of the stress resistantplant which is capable of being regenerated into a differentiated wholeplant a recombinant double stranded DNA molecule comprising:

i) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by the said sense gene;

ii) a pollen specific promoter which functions in said plant cell tocause transcription of said DNA sequence into RNA; and preferably

iii) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence.

(d) obtaining a plant cell of said stress resistant plant which has beentransformed with the gene described in step (c) above;

(e) regenerating from said transformed plant cell a plant which has beengenetically transformed with the genes described in step (a) and step(c) above and can be rendered male sterile by said chemical agent orstress; and

(f) growing said genetically transformed plant described in step (e)above in the presence of the same chemical agent or stress during pollenformation to produce a male sterile plant.

It must be understood that the preceding aspect of our invention mayalso be accomplished by transforming a plant cell with both of the genesreferred to in (a) and (c) above simultaneously, and therefore withoutan intermediate regeneration step (b).

According to another aspect of the invention as claimed herein, weprovide a method of producing hybrid seed with restored fertility fromplants selected from those species of pollen producing plants which arecapable of being genetically transformed comprising the steps of:

(a) producing a plant which carries a male sterile trait by:

i) inserting concomitantly or independently into the genome of a plantcell of said pollen producing plant which is capable of beingregenerated into a differentiated whole plant, a sense gene whichconfers on said plant resistance to a chemical agent or a naturallyoccurring or artificially induced physiological stress and a recombinantdouble stranded DNA molecule comprising:

(A) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by said sense gene;

(B) a pollen specific promoter which functions in said plant cell tocause transcription of said DNA sequence into RNA; and preferably

(C) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence;

ii) obtaining a plant cell of a plant which has been transformed withthe genes described in step (i) above;

iii) regenerating from said transformed plant cell a plant which isgenetically transformed with the genes described in step (i) above andcan be rendered male sterile by said chemical agent or stress;

(b) increasing the number of genetically transformed plants:

i) growing the genetically transformed plant described in step (a)(iii)above in isolation from the same stress or chemical agent to produce aself-fertile plant;

ii) permitting self-fertilization; and

iii) growing seed of such self-fertile plant, over a number ofgenerations in isolation from the same stress or chemical agent toincrease the number of genetically transformed plants;

(c) effecting a hybrid crossing growing said genetically transformedplants alongside plants of a suitable line of male fertile donors in thepresence of the same stress or chemical agent during pollen formation toproduce male sterile plant and to permit pollination of the male sterileplants.

In accordance with the two preceding aspects of this invention, theinvention is also directed to a plant comprising:

(A) a sense gene which confers on said plant resistance to a chemicalagent or a naturally occurring or artificially induced physiologicalstress; and

(B) a recombinant double stranded DNA molecule comprising:

i) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by said sense gene;

ii) a pollen specific promoter which functions in said plant to causetranscription of said DNA sequence into RNA; and

iii) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence, and hybrid seed containing DNAcomprising:

(a) a sense gene which confers on a plant grown from said seedresistance to a chemical agent or a naturally occurring or artificiallyinduced physiological stress; and

(b) a recombinant double stranded DNA molecule comprising:

i) a DNA sequence that codes for RNA that is complimentary to the RNAsequence encoded by said sense gene;

ii) a pollen specific promoter which functions in said plant to causetranscription of said DNA sequence into RNA; and

iii) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence.

According to another aspect of the invention as provided in the claims,we describe a method of producing a male sterile plant by transforming aplant with a recombinant DNA molecule comprising a pollen specificpromoter and a DNA sequence coding for a cytotoxic molecule. In theory,any toxic molecule which is known to be encoded by one or moreidentifiable DNA sequences may be employed within the scope of thisaspect of the invention, including possibly but not limited to ricin anddiphtheria toxin. We provide a method to produce hybrid seed withrestored male fertility by crossing said male sterile plant with asuitable male fertile plant that has been transformed with a recombinantDNA molecule comprising the pollen specific promoter and a DNA sequencewhich is in the antisense orientation to that of the DNA sequence codingfor the cytotoxic protein molecule, thereby inhibiting the expression inthe hybrid plant of said DNA sequence coding for the cytotoxic molecule.

Thus according to another aspect of the invention as claimed herein weprovide a method of producing a male sterile plant from a plant selectedfrom those species of pollen producing plants which are capable of beinggenetically transformed, which method comprises the steps of:

(a) inserting into the genome of a plant cell of said plant arecombinant double stranded DNA molecule comprising:

(i) a pollen specific promoter;

(ii) a DNA sequence that codes for a cytotoxic molecule; and

(iii) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence.

(b) obtaining a transformed plant cell; and

(c) regenerating from said transformed plant cell a geneticallytransformed plant which is male sterile.

According to yet another aspect of the invention as claimed herein, weprovide a method to produce hybrid seed with restored male fertilityfrom plants selected from those species of pollen producing plants whichare capable of being genetically transformed comprising the steps of:

(a) inserting into the genome of a plant cell of said pollen producingplant that is capable of being regenerated into a differentiated wholeplant, a gene which confers on said plant resistance to a chemical agentor physiological stress and linked to said gene a recombinant doublestranded DNA molecule comprising:

(i) a DNA sequence which codes for a cytotoxic molecule;

(ii) a pollen specific promoter which functions in said plant cell tocause transcription of said DNA sequence; and

(iii) a terminator sequence which defines a termination signal duringtranscription of such DNA sequence.

(b) obtaining a transformed plant cell;

(c) regenerating from said plant cell a genetically transformed plantwhich is male sterile;

(d) increasing the number genetically transformed plants by:

i) crossing the geneticaly transformed plant described in step (c) abovewith a suitable male fertile plant;

ii) using a chemical agent or physiological stress to eliminate plantswhich do not contain the genes described in step (a) above among plantsgrown from seed produced by such cross; and

iii) repeating such a cross over several generations with the plantsobtained as in step (d) ii) above in the presence of said chemical agentor physiological stress to increase the numbers of male sterile plants;

(e) inserting into a plant cell of suitable male fertile plant selectedfrom the same species a recombinant double stranded DNA moleculecomprising:

(i) a DNA sequence which codes for RNA that is complimentary to the RNAsequence coding for said cytotoxic molecule;

(ii) a promoter which causes transcription of the DNA sequence definedin step (d)(i) above at about the time of transcription of the DNAsequence defined in step (a)(i);

(iii) a terminator sequence which defines a termination signal duringtranscription of the DNA sequence described in step (e)(i) above;

(f) obtaining a transformed plant cell from step (d);

(g) regenerating from said transformed plant cell described in step (d)above a genetically transformed male fertile plant.

(h) producing a restorer line by permitting said genetically transformedmale fertile plant to self fertilize and growing seed of such plant,over a number of generations to increase the mumbers of geneticallytransformed male fertile plants;

(i) effecting a hybrid cross by pollinating said male sterile plantswith pollen from said genetically transformed male fertile plants.

As discussed above, in a hybrid seed production scheme where there arealternating rows of male sterile plants and male fertile plants, it maybe simpler but not essential to carry out the final selection of malesteriles in the field alongside the male fertile donors. Therefore it isdesirable if the suitable male fertile donors are previously transformedto resistance to the selective agent to avoid having to selectivelyapply said agent to the rows of male sterile plants.

In accordance with the preceding two aspects of the invention, theinvention also contemplates a male sterile plant which has incorporatedinto its DNA a recombinant DNA molecule comprising:

(i) a DNA sequence which codes for a cytotoxic molecule;

(ii) a pollen specific promoter which functions in said plant cell tocause transcription of said DNA sequence;

(iii) a terminator sequence which defines a termination signal duringtranscripton of said DNA sequence;

and hybrid seed which has incorporated into its DNA said recombinant DNAmolecule as well as another recombinant DNA molecule comprising:

(a) a DNA sequence which codes for RNA that is complimentary to an RNAsequence encoded by said gene coding for the cytotoxic molecule;

(b) a promoter which causes transcription of said DNA sequence into RNAat about the time of transcription of said gene coding for the cytotoxicmolecule; and

(c) a terminator sequence which defines a termination signal duringtranscription of said DNA sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a schematic representation of a restriction map of L 4 amicrospore specific genomic clone isolated from a Brassica napus genomiclibrary.

FIG. 1b is a schematic representation of a restriction map of L 19, amicrospore specific clone isolated from a Brassica napus genomiclibrary.

FIG. 2a is the nucleotide sequence of a portion of the clone L 4represented in FIG. 1a.

FIG. 2b is the nucleotide sequence of a portion of the clone L 19 shownin FIG. 1b.

FIG. 3 is a schematic representation of a protocol for producing thevector PAL Example 1107, discussed in greater detail below.

FIG. 4 is a schematic representation of a protocol for producing a clonecontaining a coding region of the A chain of the ricin gene.

DETAILED DESCRIPTION OF THE DRAWINGS

In FIG. 1a and 1b, the orientation of the restriction maps of themicrospore specific Brassica napus genomic clones L 4 and L 19 is from5' to 3'. Clone L 4 was used for the isolation of a microspore specificpromoter and for isolation of microspore specific gene coding fragments.Clone L 19 was used as a source for a microspore specific gene codingregion. The 5' of each clone region contains the promoter region of theclone. The shaded region of each clone demarcates the approximate codingregion of the clone. Only those restriction sites which are relevant tothe constructions detailed below are shown. The right and left arms ofthe lambda cloning vector are not shown. Indicated below each clone isthe region of DNA that has been sequenced. Those sequences are given inFIGS. 2a and 2b.

In FIGS. 2a and 2b the DNA sequence of selected portions of clones L 4and L 19, respectively, are shown. Specifically, the sequences of thedouble stranded DNA in the regions indicated in FIG. 1a and 1b are shownin the 5' to 3' orientation. For clone L 4, nucleotide 1 is 599 basepairs in front of the ATG codon of the coding region shown in FIG. 1a,the start of transcription is at nucleotide 526 and the start oftranslation is at nucleotide 600-603 (ATG). A Dde I site is shown atposition 590 immediately upstream of the ATG start codon. This Dde Isite and a further Dde I site shown 1900 nucleotides upstream of thissite were used to excise the promoter region for the construction of PAL1107. In addition, the position of the first two introns and exons areindicated. The precise start site of the third exon is not identified,only the approximate splice site is demarcated by the dashed line. The3' termination site of the gene is 3' to the last nucleotide shown inthis DNA sequence. The sequence of clone L 19 extends approximately 1.5Kb from the leftmost Eco RI site to approximately the Bgl II site in thecoding region. A Hind III site is shown at position 1899-1905. This sitecorresponds to the left most Hind III site in clone L 19.

In FIG. 3 the protocol for constructing PAL 1107 is shown. For thisconstruction, the Eco RI-Sst I fragment that encompasses most of thecoding region and 230 bases of the promoter region is subcloned into theplasmid vector pGEM 4Z using the Eco RI and Sst I sites in thepolylinker region. This clone (pPAL 0402) is digested with Eco RI andthe 2.5 Kb Eco RI fragment upstream of the coding region from thegenomic clone L 4 is added, giving the clone pPAL 0403 with areconstructed 5' region of clone L 4. The Dde I fragment is thenisolated from pPAL 0403 by gel elution, made blunt with Klenow, andcloned into pGEM 4Z previously cut with Xba I and blunted with Klenow,creating pPAL 0408. pPAL 0408 is cut with Sal I and Sst I and thepromoter fragment now containing a portion of the polylinker from pGEM4Z is then cloned into PAL 1001 previously cut with Sal I and Sst I,creating PAL 1107. The vector contains the NPT II gene for selection inplant cells, plus the promoter region from the pollen specific clone L 4followed by a portion of the polylinker from pGEM 4Z containing theseunique sites for insertion of DNA fragments to be transcribed using thefollowing restriction enzymes: Bam HI, Kpn I, Sma I and Sst I. Thepromoter region indicated is the promoter region of clone L 4, orientedin the 5' to 3' direction. DNA fragments placed at the 3' end of thisfragment will be transcribed only in pollen. T represents the 260 bp SstI-Eco RI restriction fragment containing the nopaline synthasepolyadenylation signal.

In FIG. 4 the protocol for isolating Ricin A chain sequences is shown. Aclone containing part of the ricin gene is isolated as a Eco RI fragmentin pGEM 4Z as shown. The clone is digested with Kpn I and Sst I anddigested with exonuclease III. Following SI digestion and blunt endrepair by Klenow, the clone is religated in the presence of a universaltranslation terminator (purchased from Pharmacia P. L. Biochemicals). Aclone containing this terminator and which was deleted to amino acidresidue 262 of the A chain is recovered. This clone is digested with BamHI and recloned into Bam HI cut pGEM 3 and a clone containing theorientation shown is chosen. The resultant clone is cut with Pst I andSal I, then digested with exonuclease III followed by SI and Klenowtreatment to make the DNA blunt ended. A palindromic linker thatcontains an ATG start codon and a site for Bam HI is added and the clonereligated so that a Bam HI site was introduced before the ATG start.Clones are chosen, sequenced and one in which the ATG is in frame withthe rest of the A chain coding sequence is chosen. This constructionallows the ricin A chain sequence to be excised as Bam HI fragment. Lrepresents the leader sequence for ricin whereas B represents a portionof the B chain of ricin.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It is known that there are currently a limited number of species ofplants that are capable of being transformed. Routine transformation islargely restricted to two plant families: Solanacae and Cruciferae.Examples of species included within these families are Nicotiana tabacum(Solanacae), Lycopersicon esculentum (Solanacae) and Brassica napus(Cruciferae). These include canola, tobacco and tomato plants. It isbelieved that the present invention may be carried out with plantsselected from any one of said species. Furthermore, it is fully expectedthat the present invention may be carried out with any other species ofplant that is later determined to be capable of being geneticallytransformed.

The pollen specific promoter referred to above was isolated from a plantof the species Brassica napus. It is believed, in respect of each of thespecies of plants enumerated in the preceding paragraph, each of whichwas transformed with a gene comprising said promoter, that it ispossible to use said promoter to limit the expression of a givenstructural gene sequence to microspores and to a period during pollenformation. Current published scientific literature has clearlydemonstrated that plant genes are universal and specifically that planttissue-specific promoter fragments retain their function in otherspecies. For example, wheat endosperm promoter fragments (Simpson, J.,et al, EMBO Jour. 4:2723-2729, 1985), or the Alcohol Dehydrogenase (ADH)promoter fragments (Ellis, J. G., et al, EMBO Jour. 6:11-16, 1987) canfunction properly in other plant species genetically transformed withsaid promoter fragments. Accordingly, it is fully expected that thepollen specific promoter that we have isolated is capable of beingutilized to limit the expression of any given structural gene sequenceto microspores and to a period during pollen formation, in any otherpollen-producing plant species of interest that is later found to becapable to being genetically transformed, including preferably specieswithin the families Solanacae and Cruciferae and more preferably speciesof plants selected from the genus Brassica.

According to the present invention, the following advantages areachieved over other hybridization systems:

(a) Hybrid seed production is not labour intensive and can be achievedon a large scale with commercially acceptable costs;

(b) Male sterility is simply inherited and stable in response toenvironmental stresses that limit the effectiveness ofself-incompatibility and CMS based schemes. Seed that is produced willbe relatively uncontaminated by selfed seed;

(c) The system avoids the use of defective cytoplasmic organelles thatmay themselves detract from the performance of the hybrid seed; and

(d) The system will greatly speed the development and increase thenumber of lines that can be tested as hybrid parents because it can beimposed on any inbred plant line capable of regeneration intotransformed plants without the inclusion of additional genomic DNA.Additionally, plant lines can be tested for combining ability beforeincorporation of the hybridization system.

The schemes in which male fertility is controlled by controlling thepresence of an inducer or a physiological stress are particularlyadvantageous for hybrid seed production on a large scale sincepropagation of the male sterile line can be accomplished by a simplemeans.

As a preferred embodiment of several aspects of our invention discussedabove, we provide methods of introducing genic male sterility into aplant species of the genus Brassica, in respect of which a satisfactoryCMS system is not available. These methods overcome the difficultiesassociated with the use of a CMS based male sterility system,specifically with regard to yield loss associated with defectivemitochondria.

The following terms and expressions, when used throughout the disclosureand claims herein, are to be interpreted in accordance with thedefinitions set out below unless the context dictates that said termsand expressions are not to be so limited:

Antisense DNA--A DNA sequence produced when the sense DNA is invertedrelative to its normal presentation for transcription and inserteddownstream from a promoter. The antisense DNA may be constructed in anumber of different ways, provided that it is capable of beingtranscribed into RNA which is complimentary to and capable of blockingtranslation of the RNA produced by the sense gene.

Antisense RNA--RNA that is encoded by antisense DNA.

Capable of Being Gentically Transformed--In reference to a plant, thisexpression means a plant containing cells which can stably incorporaterecombinant DNA molecule and be regenerated into a differentiated wholeplant.

Critical to Pollen Formation or Function--Any gene that is specificallyrequired for the development or function of pollen. Such genes includebut are not necessarily limited to genes which are critical tomicrospore growth and development or microspore function (eg. ability togerminate and effect fertilization) and genes which are critical andrequired for growth and developing of all cells and tissues associatedwith developing microspores such as the filament, taperum and the antherwall.,

Gene--Structural gene with flanking expression signals or sequences.

Gene That Is Critical To Cellular Function Or Development Any gene thatcodes for a product that is essential for the continued function ordevelopment of all metabolically competent cells such as but not limitedto genes involved in essential cellular structures, essentialbio-synthesis and essential metabolism.

Genomic DNA--The DNA of the plant genome.

Genomic Library--A collection of segments of genomic DNA which have beenindividually inserted into phage vectors, which collection is used toisolate specific genomic sequences.

Hybrid Plant--A plant grown from hybrid seed.

Hybrid Seed--Any seed produced by the cross-pollination of anyparticular plant inbred line by a pollen other than the pollen of thatparticular plant variety inbred line.

Microspore--A spore that develops into a pollen grain. This term is usedinterchangeably with "pollen".

Pollen Grain (or Pollen)--A structure derived from the microspore ofseed plants that develops into the male gametophyte.

Pollen Specific Promoter--A promoter that functions exclusively inpollen.

Promoter--A DNA sequence (expression signal) that causes the initiationof transcription of sequences adjacent to it.

Restriction Fragment--A specific length fragment of DNA produced by thecomplete restriction digest of a particular DNA using the specifiedrestriction digest.

Sense Gene--A DNA sequence that is capable of producing a functionalprotein product, which sequence consists of a promoter, a structuralgene and a terminator. In the context of the present invention, we shallrefer to the structural gene sought to be inactivated when combined withpromoter and terminator sequences as a sense gene.

Sense RNA--RNA that is the normal product of transcription of a sensegene and therefore capable of being translated in vitro into afunctional protein product.

Structural Gene--A DNA sequence that codes for a protein sequence.

Suitable Male Fertile Plant--Suitability for the purpose of producinghybrid seed is determined by standard crossing of different varietieswith subsequent analysis of the progeny and selection of a variety witha superior combining ability. Suitability of a male fertile plant forthe purpose of crossing with a male sterile plant to increase the numberof male sterile plants means use of, but is not necessarily limited touse of, a plant of the same inbred line from which the male sterileplant is derived. In some instances the desired increase in plants thatfunction as the male sterile female parent can be produced simply byselfing thus the suitable male fertile plant can also be itself.

Terminator--A DNA sequence (expression signal) that directs the endpoint of transcription in plants.

Transformation--The use of Agrobacterium sp. or any other suitablevector system(s) to transfer foreign DNA in a stable fashion into thegenomic DNA of a plant species.

METHODOLOGY

In the foregoing description of the invention, we set forth, in generalterms, the steps that can be employed to produce male sterile plants andhybrid seed in accordance with our invention. It is to be understoodthat these various steps may be accomplished by a variety of differentprocedures. In the following description of preferred procedures, werefer to several alternative procedures to accomplish these steps.However, it is contemplated that other variations will be apparent tothose skilled in the art. Accordingly, the scope of the presentinvention is intended to be limited only by the scope of the appendedclaims.

The isolation of genes that are critical to pollen formation may beaccomplished by a variety of procedures. In accordance with one aspectof the method of our invention, we identify by known methods, genes thatare only expressed at specific stages during pollen development whoseregulation is tightly controlled. The genes may be isolated by cloningtechniques in accordance with the detailed method set out below. Theisolation step may also be accomplished by ascertaining, according tostandard cloning techniques discussed below, whether a given gene whichis known to be critical to pollen formation and expressed exclusively inmicrospore tissue in one plant, has the same utility in another plant.It is also known that certain genes are critical to cellular functionand are expressed in all cell types. These genes may be isolated usingthe published DNA sequences for these essential genes, some of which areconserved amongst the plant and animal species, and in conjunction witha promoter that limits gene expression to only pollen tissue be used forthe construction of an antisense RNA gene that caused male sterility.

The antisense genes may be constructed according to any one of a varietyof known methods. The preferred method of construction detailed below isto excise the double stranded coding region of the sense gene or afunctional fragment thereof and to insert said double stranded DNAmolecule downstream from a promoter, in an inverted orientation relativeto its normal presentation for transcription. A terminator structure ispreferably added to the end of this antisense gene. It is possiblewithin the scope of the present invention to synthetically produce saidantisense DNA sequence or a functional fragment thereof, according toknown methods, and insert said sequence by known methods downstream froma promoter that will cause timely transcription of same into sufficientquantities of RNA.

The antisense gene may be introduced into the plant cell by any one of avariety of known methods preferably by first inserting said gene into asuitable vector and then using said vector to introduce said gene into aplant cell. The transformed plant cell is selected for by the presenceof a marker gene for any one of a variety of selectable agents which iscapable of conferring resistance to transformed plant cells to sameagent. Transformed plant cells thus selected for can be induced todifferentiate into mature plant structures. Additionally, it is to beunderstood that whereas some aspects of this invention may require thetransformation of a plant cell with two different genes, that thesegenes may be physically linked by both being contained on the samevector or physically seperate on different vectors. It is alsounderstood that if the genes are on different vectors, that thetransformation of a plant cell can take place with both vectorssimultaneously providing each vector has a unique selectable marker.Alternatively, the transformation of a plant cell with the two vectorscan be accomplished by an intermediate regeneration step aftertransformation with the first vector.

Where the cost is warranted, and maintenance cannot be readilyaccomplished as discussed above, transformed plant cells can be grown inculture according to routine methodology to produce a cell linecomprising a large number of transformed cells. A large number oftransformed plants can be regenerated according to routine methodologyfrom said transformed plant cell line to increase and maintain the malesterile line. Routine methodology for culturing such cells andregenerating transformed plants from such cells is described in suchplant tissue culture hand books as: Plant Tissue and Cell Culture, C. E.Green, D. A. Somers, W. P. Hackett and D. D. Biesboer, Eds., Alan R.Liss, Inc., New York, Experiments in Plant Tissue Culture, Dodds, J. H.and Roberts, L. W. Eds., 1985, Cambridge University Press, or CellStructure and Somatic Cell Genetics of Plants, Vasil, I. K., Ed., 1984,Academic Press, Handbook of Plant Cell Culture, Volume 4, Techniques andApplications, Evans, D. A., Sharp, W. R., and Ammirato, P. V., Eds.,1986, Macmillan Publishing Company.

It is also possible to produce male sterile plants by fusing cells ofthe transformed plant cell line with cells of plant species that cannotbe transformed by standard methods. A fusion plant cell line is obtainedwhich carries a genetic component from both plant cells. The fusioncells can be selected for cells that carry the antisense gene and inmany cases induced to regenerate into mature plants that carry the malesterile trait.

It is to be understood that any one of a number of different promoterscan be used to regulate the expression of the antisense gene, providedthat the promoter causes transcription of said antisense gene at theproper time and into sufficient quantities of RNA to block the functionof the sense RNA and thereby prevent its translation into proteinrequired for proper microspore development. Complete inhibition of theexpression of these genes is not needed, only expression that is reducedto the point that normal pollen development is interfered with. It ispossible to use a combination of different genes and promoter structuresto interfere with normal pollen development. A promoter or a methodwhich could be used to amplify the expression of the antisense genecould be useful to ensure adequate production of antisense RNA.

When using a pollen specific promoter to inactivate a sense gene that iscritical to pollen formation or function or to cellular growth ordevelopment, we discussed that it is often difficult to determine, apriori, what pollen specific promoter will effectively block thefunction of that gene. We discussed that it is preferable to use apollen specific promoter that displays a similar developmental patternto that gene. A convenient method to determine when the sense genesought to be inactivated is expressed is to isolate RNA from developingmicrospores at different stages and to analyze this RNA for theexpression of the sense gene by the so-called Northern analysis. Thisprocess will allow for the determination of the exact developmentalperiod in which the sense gene is expressed. In order to determine theperiod in pollen development in which the pollen specific promotersought to be used to activate said sense gene is expressed, a similarseries of analysis can be carried out using as a probe for theexpression of said pollen specific promoter a reporter gene joined tosaid promoter or the naturally occurring sense gene under the control ofthe pollen specific promoter found in the plant originally used for theisolation of the sense gene. When the pollen specific promoter isisolated from one plant and used in a different plant species thepreferred method is the use of a reporter gene joined to said promoterto determine the exact developmental timing that that promoter fragmenthas in that particular plant species.

It is also to be understood that the antisense DNA does not necessarilyhave to code for RNA that is complimentary to the entire mRNA chainencoded by the sense gene provided that the mRNA encoded by theantisense gene is otherwise capable of hybridizing with and blockingtranslation of the native mRNA species targeted for inactivation.Accordingly the term antisense DNA when used in disclosure claims hereinencompasses a functional fragment thereof.

A. Isolation of Genes that are Critical to Proper Development ofMicrospores

To isolate genes that are specifically expressed in developingmicrospores, a genomic library of plant DNA may be constructed from DNAisolated from fresh young leaf material according to standardmethodology, described in Molecular Cloning, a Laboratory Manual(Maniatis, T. Fritsch, E. F., and Sambrooks, J., Cold Spring HarbourLaboratory, Cold Spring Harbour, New York, 1982) and screened withprobes derived from several tissues, one of which is made frommicrospore RNA. The other probes should be made from RNA from differenttissues so as to represent genes expressed in tissues of the plant thatwould not be expected to include genes that are expressed inmicrospores. Examples of these would include but are not limited to suchtissues as leaf, roots, seeds, stigma, stem and other plant organs.However, some genes will be expressed in all tissues. By surveying manyplant tissues, it is possible to isolate genes expressed exclusively inmicrospores.

The microspore RNA may be isolated from microspores that are at theearly to late uninucleate stage. Though it is possible to use othermicrospore stages certain difficulties might be confronted. The use ofpremeiotic stage microspores could prove to be problematic in many plantspecies since in some plants the callose wall has not formed yet andisolation of the immature microspores is technically difficult.Microspores that are isolated at the stages post nuclear division mayhave limited nuclear gene activity when compared to earlier stages.Therefore, the early to late uninucleate stages are preferred.

The microspores may be conveniently isolated from the anthers by manualdissection of the buds from the growing plant and subsequent removal ofthe anthers. Microspores are isolated from the anthers by gentlegrinding of the anthers in a mortar and pestle in the presence of asolution of 10% sucrose. The extract is then filtered through a 44 umnylon mesh and the microspores are collected from the filtrate bycentrifugation at 3000×g for one minute. The pelleted microspores areresuspended in 10% sucrose, filtered and pelleted as before. Othermethods of isolation can also be used to obtain microspores.

Tissues other than microspores can be disrupted by a variety of methodsand the disrupted tissue can be used for RNA extraction. It isconvenient to disrupt the tissue by using a motor driven homogenizerwith 10 mls of a solution of 6M guanidinium-HCl, 0.1M Na acetate, pH6.0, 0.1M beta-mercaptoethanol per gram of tissue. The homogenate iscentrifuged at 5000×g and the cleared supernatant is layered over asolution of 6M CsCl in Tris-EDTA buffer (TE buffer). Centrifugation at100,000×g for 12-20 hours at 15°^(C) is used to pellet the RNA which issubsequently resuspended in water and reprecipitated in the presence of0.3M Na acetate and 2 volumes of ethanol. RNA is recovered bycentrifugation and resuspended in water. The RNA obtained from suchmethod can be fractionated by oligo-d-T cellulose chromatography toseparate the polyadenyiated mRNA from the bulk of the non-polyadenylatedRNA. The microspore RNA is conveniently isolated by using a tightfitting motor driven glass homogenizer to disrupt the microspores. Thehomogenization of the microspores, 1 ml of a solution of 6Mguanidinium-HCl, 0.1M Na acetate, pH 6.0, 0.1M beta-mercaptoethanol per300 ul of packed microspores used as a RNA extraction buffer during thedisruption of the microspores. The homogenate is centrifuged at 5000×gand the cleared supernatant is layered over a solution of 6M CsCl in TEbuffer. An overnight centrifugation at 100,000×g is used to pellet theRNA which is subsequently resuspended in water and reprecipitated in thepresence of 0.3M Na acetate and 2 volumes of ethanol. Other methods ofRNA extraction can be used to obtain the RNA from the tissues described.Standard methodology using oligo-dT cellulose is used to obtainpolyadenylated mRNA from these total RNA preparations.

The mRNA is labelled for the purpose of detection. It is convenient tomake radioactive cDNA by using said mRNA and AMV reverse transcriptasein the presence of random hexanucleotide primers and alpha- ³² P!-dCTP.Probes are used for hybridization to nitrocellulose plaque lifts ofplates containing the clones of the genomic library. Clones that can beidentified as strongly hybridizing only to microspore cDNA and not cDNAfrom any other tissue examined are chosen. These clones are plaquepurified and grown for DNA isolation. Alternative techniques formanipulating DNA and RNA as well as recombinant DNA, growing andisolating clones can be found in standard laboratory manuals, such asMolecular Cloning, A Laboratory Manual (Maniatis, T., Fritsch, E. F.,and Sambrook, J. Cold Spring Harbour Laboratory, Cold Spring Harbour,N.Y., 1982).

In the case where the genomic DNA sequence of L 4 or L 19 from Brassicanapus are used to carry out certain aspects of this invention, thepreferred method to obtain ("isolate") a sense gene that is critical topollen formation or function is to synthetically produce a homologousDNA sequence according to standard methodology (see for example Gait, M.J. (1984) in Oligonucleotide synthesis, a practical approach Gait, M. J.ed., pp 1-22 IRL Press, Oxford, U.K.), label said sequence for thepurpose of detection and use said labelled sequence to screen a Brassicanapus genomic library produced according to the methods described.

B. Construction of Antisense Genes

The identity of the promoter and coding region of a given genomic cloneis determined by restriction mapping and hybridization analysis. Thismay be accomplished by hybridization of cDNA probes made from microsporeRNA with restriction fragments of said DNA clones immobilized onnitrocellulose. Restriction endonuclease fragments which contain boththe coding region and the regions of DNA on either side of the codingregion are isolated by sub-cloning in the appropriate vectors. Onceisolated, it is convenient to use techniques such as SI mapping and DNAsequencing to obtain exact coding regions and restrictions sites withinthe subcloned DNA. This analysis is easily accomplished once thepolarity with respect to gene transcription is known.

In order to determine the polarity of transcription of the sense geneindividual restriction fragments may be subcloned in commerciallyavailable vectors such as pGEM3, pGEM4, or pGEM3Z, pGEM4Z (availablefrom Promega Biotech, Madison, Wisc., U.S.A.). By using these vectorsone is able to generate single stranded KNA probes which arecomplimentary to one or the other strands of the DNA duplex in a givensubclone. These strand specific probes are hybridized to mRNA, in orderto establish the polarity of transcription. Among these probes, one canisolate those probes which hybridize with and hence are complimentary tothe mRNA. Using this information it is possible to clearly determinefrom what DNA strand of the double strand genomic DNA molecule the sensemRNA has been transcribed.

In order to isolate promoter DNA sequences from the coding region, thepGEM series of vectors can be used for the unidirectional deletion ofsequences from the individual subclones. Additionally, transcriptionalstart sites of promoter regions may be mapped. This may be convenientlyaccomplished by using a single stranded RNA probe transcribed from theindividual subclones in hybridization-protection experiments. Detaileddescriptions of these experimental protocols can be found in a number oflaboratory handbooks and in the manufacturer's technical notes suppliedwith the pGEM series of vectors. These experiments will clearlyestablish the promoter and coding regions of the pollen specific genomicclones. The sequence of individual deletions in the pGEM vectors can bedetermined by didoexy sequencing of plasmid mini-preps as described inthe manufacturer's technical notes. Deletion subclones that are deletedto very near the start of transcription or specific restrictionfragments that encompass the promoter region or the promoter region andstart of transcription are chosen for the construction of genes that areexpressed only in developing microspores of pollen bearing plants.Usually the promoter fragment is inserted upstream of a terminator suchas the nos terminator found in pRAJ-221 (available from ClonetechLaboratories, Palo Alto Cailf.) and specific restriction fragments whichare to be transcribed into antisense RNA are inserted between thepromoter and terminator sequences. The entire construct is verified bycombination of sequencing and restriction digests. The antisense genethus constructed and verified may be inserted in T-DNA based vectors forplant cell transformation. T-DNA vectors that contain a selectablemarker are preferred. It is to be understood that the antisense gene canbe constructed in a variety of ways depending on the choice of vectors,restriction enzymes and the individual genes used. For example, it maybe convenient as demonstrated by Example 1, to insert restrictionfragments intended to be transcribed into antisense RNA into a T-DNAbased vector to which a promoter and terminator structure have beenpreviously added. Alternatively, it is possible to insert a promoterfragment upstream of a coding region and terminator that has beenpreviously added to a T-DNA based vector. In addition, it may bedesirable in some crops not to insert the antisense gene into a T-DNAbased vector but rather into a vector suitable for direct DNA uptake.Promoters other than microspore specific promoters can be used andjoined with specific restriction fragments of genes and terminatorsprovided that these promoters function in microspores.

C. Transformation of Plant Cells

A number of published articles have dealt with the subject of planttransformation. Generally speaking, two types of methodology exist fortransformation of plant cells: (1) use of an infectious agent such asAgrobacterium or viruses to deliver foreign DNA to plant cells, and (2)mechanical means such as naked DNA uptake or electropotation. In bothcases, the desired result is the uptake of foreign DNA into the plantcell and subsequent stable integration of the foreign DNA into thenuclear genetic component of the plant cell. In the ensuing examples, wedescribe a particular type of methodology that may be used to producetransformed plants. However, it is to be understood that other methodscan be used for the purpose of production of transformed plantscontaining antisense genes. These include but are not limited to:protoplast transformation, transformation of microspores, whole plantwounding with Agrobacterium followed by recovery of and regenerationfrom infected tissue, naked DNA uptake with Agrobacterium deliverysystems and other methods such as electroporation.

D. Regeneration of Plants from Transformed Plant Cells

After transformation of a plant cell, the plant cell is allowed to growand develop into a whole plant. Usually this takes place over a periodof time during which time selection of the transformed plant cells isaccomplished by the application of a selective pressure such as anantibiotic, drug or metabolites that are toxic to the plant cell.Resistance to the selective pressure is conferred on the plant cell bythe presence of a resistance gene on the transformation vector thusallowing the transformed plant cell to grow in the presence of theselective pressure.

In some cases, adjustment of the growth regulating substances isrequired in order for the plant cell to differentiate into a matureplant. This may involve a number of steps in which the transformed plantcell is allowed to grow into an undifferentiated mass commonly referredto as a callus. This callus is then transferred to a medium which allowsfor the differentiation of said callus into organized plant structuresand eventually mature plants. Alternatively, some procedures may involvethe differentiation of the transformed plant cell directly into astructure such as a shoot which is then removed to media that allows forrooting and subsequent growth and flowering. For each individual plantspecies the choice of steps is determined experimentally.

E. Testing for the Presence and Expression of Antisense and Marker Genes

Plants which are regenerated from transformed plant cells are tested forthe expression of the marker gene which is usually the gene that confersresistance to the selective agent. In the case of the commonly used geneneomycin phosphotransferase (NPT II) which confers resistance tokanamycin and the antibiotic G-418, gene expression is tested for by invitro phosphorylation of kanamycin using techniques described in theavailable literature or by testing for the presence of the mRNA codingfor the NPT II gene by northern blot analysis of RNA from the tissue ofthe transformed plant. Expression of the antisense gene is monitoredusing the same northern blot techniques. Single stranded RNA probeswhich are either homologous to sense or antisense RNA transcripts areused to detect said transcripts in developing microspores such that theexpression of the sense and antisense gene may be ascertained. It ispreferable to use agarose gel electrophoresis to separate transcriptsfrom the sense and antisense genes according to size and to do so underdenaturating conditions. In the case where microspore specific geneexpression of antisense genes is sought to be accomplished it isadvisable to test for the expression of antisense genes in microsporesand tissues other than microspores such as leaves, roots, etc. so thattissue specific gene expression of antisense genes in microspores can beverified.

The presence of a stably integrated sense or antisense gene in thegenetic component of the plant cell may also be ascertained by using theso-called southern blot techniques. In this procedure, total cellular ornuclear DNA is isolated from the transformed plant or plant cell andpreferably restricted with a restriction enzyme that usually cuts thevector used for transformation at discreet sites, thereby giving rise todiscreet fragments. These discreet vector fragments can be detected inthe nuclear or total DNA of the transformed plant or plant cells byemploying standard gel electrophoresis and hybridization techniques.

Formation of microspores in plants which contain the antisense genes ismonitored first by visual and microscopic examination of the antherstructures. As maturation of the flower structure occurs, antherformation is expected to be delayed or completely inhibited such that nomature pollen grains are formed or released.

As the activity and hence effectiveness of any introduced recombinantDNA molecule is influenced by chance by the position of insertion ofsaid molecule into the plant DNA the degree to which the insertedrecombinant DNA may cause or render the plant sensitive to agents thatcause reduced male fertility may vary from plant to plant. Accordingly,it will be necessary to select a plant which produces no functionalpollen grains.

F. Hybrid Seed Production

Production of hybrid seed is accomplished by pollination of transformedmale sterile plants with pollen derived from selected male fertileplants. Pollination can be by any means, including but not limited tohand, wind or insect pollination, or mechanical contact between the malefertile and male sterile plant. For production of hybrid seeds on acommercial scale in most plant species pollination by wind and by insectare preferred methods of pollination. Selection of plants for pollendonation is determined by standard crossing of different plants withsubsequent analysis of the progeny and selection of the lines with thebest combining ability. Restoration of fertility in the hybrid seed is,where warranted, accomplished by using the methodology detailed in thespecific examples.

The invention is illustrated but not limited by the following examples:

EXAMPLE 1

This example relates to the isolation of a pollen specific promoter anda coding sequence (clone L 4) which is critical to pollen formation andexpressed only in developing microspores. The promoter and codingsequence are isolated from, Brassica napus cv. Westar. The antisense DNAis placed under the control of the same polen specific promoter and usedto transform plant cells derived from a plant of the species Brassicanapus.

Two microspore specific genomic clones, L 4 and L 19, were isolated froma genomic library constructed from a plant of the species Brassica napuscv. Westar by screening said library with probes constructed from RNAisolated from microspores, leaves, seeds and stems according to themethods specified above. The microspores were isolated from flower budsthat were 3 to 5 mm in length.

Restriction maps of these two clones are shown in FIG. 1a and 1b. The 5'and 3' regions as well as the coding regions of these clones are shownand were determined by hybridization analysis according to the methodsdescribed above. The clone L 4 consists of two contiguous Eco RIrestriction fragments of 2.5 and 5.7 Kb. Clone L 19 consists of a single10.5 Kb Eco RI fragment. Not all restriction sites are shown in FIGS. 1aand 1b, only those relevant to the individual constructs detailed below.

The regions of the clones for which the nucleotide sequence has beendetermined are also indicated in FIGS. 1a and 1b. These nucleotidesequences are shown in FIGS. 2a and 2b, respectively. A DNA fragmentthat functions as a microspore specific promoter in transgenic plantswas isolated from L 4 and consists of nucleotides 1-595 of the DNAsequence shown for L 4, in FIG. 2a. The ability of this DNA fragment tofunction as a microspore specific promoter in transgenic plants wasdetermined by insertion of the entire 5.7 Kb Eco RI DNA segment of cloneL 4 into tobacco plants and analyzing different tissue for theexpression of the coding region of clone L 4 under high stringencyconditions that allow for the specific detection of expressed codingsequence of clone L 4. Under these conditions, mRNA transcribed from theL 4 inserted DNA was only detectable in early uninucleate microsporesisolated from the transgenic tobacco, and not in any other tissueexamined which included stems, leaves, flower petals, filaments, stigmaand stamen, large mature anthers. This particular inserted DNA segmentcontains the entire coding region of clone L 4 and 230 bases of 5'promoter sequence. This length of DNA represents the minimal amount ofpromoter DNA sequence needed to retain microspore specific promoteractivity in transgenic tobacco. Provision of additional 5' promotersequence does not alter the specificity of this DNA in terms of itsfunctioning as a pollen specific promoter, it only increases the levelsof pollen specific expression. Therefore, in order to maximize thelevels of tissue specific gene expression from this specific DNAsequence, additional upstream region of the 5' region of clone L 4 wasincluded in the DNA constructions listed below. A DNA fragmentcontaining additional 5' sequences of the promoter region contained inclone L 4 was isolated as a Dde I fragment. This 1.9 Kb fragment wasisolated in the following fashion: The Eco RI-Sst I fragment thatencompasses the 5' region of the coding sequence and 235 nucleotides ofthe promoter were subcloned into the commercially available vectorpGEM-4Z (sold by Promega Biotech Madison, Wisc., U.S.A.) using the EcoRI and Sst I sites in the polylinker region. The resultant plasmid wasnamed pPAL 0402. pPAL 0402 was digested with Eco RI and the 2.5 Kb EcoRI fragment of clone L 4 was ligated into this site, creating a plasmidthat reconstructed the 5' region of the genomic clone L 4. This plasmidwas named pPAL 0403. pPAL 0403 was digested with Dde I and the 1.9 KbDde I fragment that encompasses the promoter region of clone L 4 wasisolated by gel elution, made blunt ended with Klenow fragment andsubcloned into the Xba 1 site of pGEM-4Z previously made flush withKlenow treatment. Two orientations were obtained, the one shown in FIG.3 was chosen for further constructions and was named pPAL 0408. Thispromoter fragment in pPAL 0408 contains the DNA sequences required forlimiting the expression of any gene adjacent to the 3' end of thisfragment solely to developing microspores. At the extreme 3' end of thisfragment is the start of transcription site so that any DNA sequenceplaced at this end of the fragment will be transcribed and will containa 69 (+or -2 bp) untranslated leader sequence without any ATG initiationcodon. A cassette transformation vector using this promoter fragment wasconstructed using the binary transformation vector BIN 19 (obtained fromthe Plant Breeding Institute and described in Bevan, M., 1984, Nucl.Acids Res. 12:8711-8721) and adding to BIN 19 the nopaline synthasepolyadenylation signal (nos ter) isolated as a Sst I-Eco RI 260 bprestriction fragment from the plasmid pRAJ 221 (available from ClonetechLaboratories, Palo Alto, Calif., USA) and inserting this nos terfragment into the Sst I-Eco RI restriction sites of BIN 19. Theresultant plasmid vector was called PAL 1001. The promoter from pPAL0408 was added to PAL 1001 by cutting pPAL 0408 with Sal I and Sst I andcloning this promoter-polylinker containing fragment in the vector PAL1001 using the Sal 1 and Sst I sites of PAL 1001. A binarytransformation vector PAL 1107 was constructed.

The details of the construction of PAL 1107 are shown in FIG. 3. Thisvector has (in a 5' to 3' order) the promoter from clone L 4, followedby a portion of the polylinker from PGEM-4Z containing the followingrestriction sites: Bam HI, Sma I, Kpn I and Sst I followed then by thenos ter. This vector allows for the convenient insertion of DNAfragments for transcription under the control of the pollen specificpromoter isolated from clone L 4.

To this PAL 1107 vector was added a coding region fragment of clone L 4in the antisense orientation by digesting pPAL 0402 with Bam HI and HincII and ligating this 1.68 KG coding region restriction fragment to BamHI-Sma 1 cut PAL 1107. This construction was mobilized intoAgrobacterium tumefaciens. The Agrobacterium strain GV 3101 carrying theTi plasmid pMP 90 (Koncz, C. and Schell, J. 1986, Mol. Gen. Genet.204:383-396) to provide the vir functions in trans was used as arecipient this binary vector (PAL 1107 containing the antisenserestriction fragment) by delivery of the binary vector throughtripartite mating and kanamycin selection on minimal media. Binaryvectors allowed for the selection of transformed plants by the virtue ofcarrying the NPT II (neomycin phosphotransferase) gene under the controlof the nos promoter providing for resistance to kanamycin and G418 intransformed plant cells.

Transformation of Brassica napus plants with this antisense gene wasaccomplished by cocultivation of thin epidermal layers from stems ofBrassica napus, cv. Westar. The cocultivation was performed usingsurface sterilized stem epidermal layer peels as follows. The upperthree internodes of the stem of plants that had fully developed budclusters but whose buds had not yet opened (but were within 1-3 days ofdoing so) were surface sterilized by rinsing in 70% ethanol for 5 to 6seconds followed by 2% sodium hypochlorate for 10 minutes and then threetimes in sterile distilled water. Segments were cultured for 1 day onmodified MS media in which NH₄ NO₃ was replaced with 60 mM KNO₃ andhaving in addition B5 vitamins with 40 mg per 1. of FeEDTA as a sourceof iron, 0.5 mg per 1--naphthalene acetic acid and 10 mg per 1 benzyladenine with 3% sucrose and 0.8% agar, pH 5.8. This modified medium ishereinafter referred to as EPL. All cultures were maintained undercontinuous light (approximately 70 uE per sq.m per sec). After thisperiod of time, the explants were removed from the surface of theculture media and exposed to the Agrobacterium strain carrying PAL 1107into which the 1.68 Kb Bam HII-Hinc III fragment of L 4 was inserted.This was done by using sterile forceps to place the explant in contactfor a few moments with a confluent layer of bacteria that was growing onthe surface of a petri plate. The bacteria were grown in minimal mediacontaining kanamycin at 100 ugs. per ml. It is also possible to exposefor a few seconds the epidermal layers to the bacteria in liquid cultureliquid media such as LB-MG (Beringer, J. E., 1974, Jour, Gen. Microbiol.84: 188-198) containing 100 ugs. per ml. kanamycin and 20 uMacetosyringone!, with similar results. The epidermal layers were blottedon sterile filter paper and placed on plates that contained EPL. Theseplates were covered with a layer of tobacco cells (the feeder layer)from a cell suspension of Nicotiana debneyi and overlayed with sterilefilter paper upon which the epidermal layers were placed. After amaximum of three days of cocultivation with the Agrobacterium, thesegments were transferred to EPL media without a feeder layer or filterpaper. This media contained in addition to the normal components, 100 ugper ml of kanamycin sulfate for selection of transformed plant cells and500 ug per ml of cefotaxamine to kill the Agrobacterium bacteria.

Shoots which were regenerated from the epidermal layers weresubsequently tested for the activity of the NPT II enzyme. The shootsthat tested positive for-the NPT II enzyme were rooted on B5 mediacontaining 2% sucrose, 0.8% agar and 0.5 mg per 1. of bothindole-3-acetic acid and -naphthalene acetic acid. After rooting, theplants were transferred to soil and placed in a misting chamber for 7days. The plants were then transferred to a growth chamber where theywere allowed to develop and flower.

Southern blot analysis of DNA taken from these plants confirmed thepresence of the antisense gene.

EXAMPLE 2

In this example we use the same promoter as in example 1 and a differentpollen specific sense coding sequence (clone L 19) to construct theantisense gene and transform plant cells derived from a plant of thespecies Brassica napus.

To the vector PAL 1107 wa added a 1.3 Kb Hind III restriction fragmentcontaining coding sequence from clone L 19. This fragment was first madeblunt by Klenow treatment and this blunt ended fragment was cloned intohe unique Sma 1 site of PAL 1107. Clones containing this 1.3 Kb Hind IIIfragment in the antisense orientation were chosen and used for thetransformation of Brassica napus stem epidermal layer peels as describedabove.

EXAMPLE 3

In this example, we transform tobacco which was previously transformedto hygromycin resistance with and antisense that blocks the hygromycinresistance gene under the control of a pollen specific promoter derivedfrom Brassica napus (clone L 4).

The 0.8 Kb Bam HI restriction endonuclease fragment encoding thehygromycin phosphotransferase gene (Gritz and Davies, 1983 Gene25:179-185) was isolated from the plasmid pVU1011 supplied by S.Scofield of the Plant Breeding Institute, Cambridge, U.K. pVU 1011 isBIN 19 into which has been added a CaMV 35S promoter controlling theexpression of the hygromycin phosphotransferase gene followed by the nosterminator. The 0.8 Kb Bam HI restriction endonuclease fragment wasligated into the single Bam HI restriction endonuclease site in PAL 1107previously described in example 1. Clones containing the 0.8 kbhygromycin phosphotransferase gene fragment in PAL 1107 were restrictionmapped and one which contained said fragment in the antisenseorientation was isolated and named PAL 1107HYGAS.

This vector was used for the production of male sterile plants bytransformation of a plant that had been previously transformed tohygromycin resistance with a vector called pGUS-HYG. We now turn to theconstruction of pGUS-HYG. pGUS-HYG is pVU1011 in which the NPT II gene(neomycin phosphotransferase gene) is inactivated by insertion of a DNAfragment from pRAJ 221 into this NPT II gene. Since the intact pVU1011vector confers hygromycin resistance in addition to kanamycin resistancewe inactivated the NPT II gene in order to produce a vector whichconfers only hygromycin resistance. To insert a DNA fragment into theNPT II gene the pVU1011 plasmid was partially digested with Sph Irestriction endonuclease. The cut pVU1011 was made blunt ended by theuse of Klenow fragment. The DNA fragment from pRAJ 221, which containsthe CaMV 35S promoter controlling the expression of the GUS(beta-glucuronidase) gene followed by the nos terminator, was isolatedby restricting pRAJ 221 with Hind III and Eco RI restrictionendonucleases and making this fragment blunt ended with Klenow fragment.Said fragment was then ligated to the partially digested blunt endedpVU1011. Clones containing the GUS gene in the middle of the NPT II genein pVU1011 were identified by restriction endonuclease analysis. Such aclone confers resistance in the plant only to hygromycin and not tokanamycin or G418 by virtue of the fact that the NPT II gene isinterrupted by the presence of the GUS gene. The inserted GUS geneprovides a convenient expression marker for plant transformation. Thisclone was named pGUS-HYG.

We now turn to the transformation of tobacco leaf discs with the vectorspGUS-HYG and PAL 1107HYGAS. Both pGUS-HYG and PAL 1107HYGAS weremobilized into Agrobacterium tumifaciens GV 3101 containing pMP-90 toprovide vir functions in trans by tripartite mating and selection onminimal media. Leaf discs were excised from leaves which were mediumgreen and less than 8 inches long and were surface sterilized byexposure to ethanol for 5 to 6 seconds and subsequent exposure to a 1%solution of sodium hypochlorite for a few minutes or until the cut edgeof the petiole turned white. Leaves were rinsed with sterile distilledwater. Discs approximately 0.5 to 0.7 cm large were excised from theleaves with a sterile cork borer. The leaf discs were placed on mediaconsisting of 0.8% agar, MS salts, B5 vitamins, 3% sucrose, 1 mg per 1.of benzyl adenine and 0.1 mg per 1. of -naphthalene acetic acid. Discswere placed upper epidermis side down on this media. Cultures weremaintained on a 16 hour photoperiod at 25° C. After one day of culture,discs were removed and placed in a 10 ml overnight culture ofAgrobacterium containing pGUS-HYG. The discs and bacteria were gentlyshaken for a few moments to insure bacterial contact with the leafdiscs. The leaf discs were removed and blotted dry on sterile filterpaper and placed on new media. This media contained cefotaxamine at 500ugs. per ml., in addition to the ingredients of the first culture media,in order to kill the bacteria and also hygromycin at 50 ugs. per ml. forselection of transformed plant cells that carried the hygromycinphosphotransferase gene. Shoots were allowed to regenerate on thisselective media from these explants. After 3 to 8 weeks coculture onselective media, shoots were large enough to be transferred to rootingmedia. Plants were rooted on B5 media that contained: 0.8% agar, 2%sucrose and 0.5 mg per ml. of both naphthalene acetic acid and indoleacetic acid. After rooting, plants were transferred to soil and kept ina misting chamber for 7 days and subsequently transferred to greenhousegrowth facilities. Plants were fertilized weekly and watered daily untilmaturity. Transformed tobacco containing the sense hygromycin gene frompGUS-HYG was re-transformed with PAL 1107HYGAS using the leaf discprocedure described above. Retransformed plant cells were exposed tohygromycin at 50 ugs. per ml. and kanamycin at 300 ugs. per ml. toselect for the presence of both the hygromycin phosphotransferase genefrom pGUS-HYG and the NPT II gene contained in PAL 1107HYGAS.Regenerated plants were obtained from said cells that were resistant toboth antibiotics and were grown in the presence of 50 ugs. per ml. ofhygromycin by rooting in one-tenth strength MS salts in 0.8% agarcontaining hygromycin. Optionally it is possible to do a doubletransformation using both PAL 1107HYGAS and pGUS-HYG, by coculturing theplant explants with both bacteria containing each vector and selectingfor doubly transformed cells using both kanamycin and hygromycin in themedia.

EXAMPLE 4

In this example we repeat the procedure used in example 3 with Brassicanapus.

The vectors described in example 3 were also used for the production ofmale sterile plants in Brassica napus. The vectors PAL 1107 HYGAS andpGUS-HYG were used for transformation of thin stem epidermal layers ofBrassica napus, cv. Westar stems. The cocultivation was performed usingsurface sterilized stem epidermal layer peels as described in example 1.The vector pGUS-HYG was used for an initial transformation, then plantsresistant to hygromycin were recovered.

For the production of male sterile plants in the plasmid PAL 1107 HYGASwas used to retransform Brassica napus stem epidermal peels from plantsthat had been previously transformed to hygromycin resistance using thehygromycin phosphotransferase gene in pGUS-HYG.

Retransformed Brassica napus plant cells containing the sense hygromycingene from pGUS-HYG and the antisense hygromycin gene in PAL 1107 HYGASwere exposed to hygromycin at 10 ugs. per ml. and kanamycin at 100 ugs.per ml. to select for the presence of both the hygromycinphosphotransferase gene from pGUS-HYG and the NPT II gene contained inPAL 1107HYGAS. Regenerated plants were obtained from said cells thatwere resistant to both antibiotics.

EXAMPLE 5

In this example, we repeat the procedure used in example 3 with tomato.

The vectors described in example 3 were used for the production of malesterile plants in tomato, Lycopersicon esculentum. The vectors PAL1107HYGAS and pGUS-HYG were used. Each vector was used individually fortransformation by first mobilizing the vector into Agrobacteriumtumefaciens LBA 4404 via triparental mating (Bevan, M., 1984, Nucl.Acids Res. 12:8711-8721). Tomato plants resistant to hygromycin werefirst obtained by using pGUS-HYG to trans#orm tomato leaf discsaccording to published procedures (Horsch et al, 1985 Science227:1229-1231). According to this method, leaves are surface sterilizedby rinsing with 70% ethanol for a few moments followed by soaking in asolution of 1% sodium hypochlorite for approximately 10 minutes or untilthe edge of the leaf bleaches and finally rinsing in sterile water.Discs are excised from the leaf with a sterile cork borer and incubatedfor one minute with gentle shaking in a solution of Agrobacteriumcontaining the transformation vector grown overnight in standardbacterial media (LB) at pH 5.6. The discs were blotted dry and placed ona feeder layer as described in example 1 that were present on a mediacontaining MS based salts, 0.8% agar, 3 mg per ml. benzyladenine and 0.3mg. per ml. indole-3-acetic acid for a period of three days. After thistime, discs were transferred to the same media without a feeder layerand containing in addition to the normal components, 500 ugs.. per ml.cefotaxamine and 100 ugs. per ml. kanamycin for vectors containingkanamycin resistance as a selectable marker. For vectors which conferhygromycin resistance, kanamycin was omitted and 10 ugs. per ml. ofhygromycin was used. Shoots which regenerated were transferred torooting media (B5 based salts, 0.5 mg./per 1 indole-3-acetic acid,and-napthalene acetic acid with 0.8% agar) and allowed to develop intomature plants.

For the production of male sterile plants, PAL 1107HYGAS was used tore-transform leaf tissue taken from tomato plants that were previouslytransformed to hygromycin resistance using the hygromycinphosphotransferase gene in pGUS-HYG. Retransformed tomato plant cellscontaining the sense hygromycin gene from pGUS-HYG and the antisensehygromycin gene from PAL 1107HYGAS were selected for with hygromycin at10 ugs. per ml. and kanamycin at 100 ugs. per ml. to select for thepresence of both the hygromycin phosphotransferase gene from pGUS-HYGand NPT II gene contained in PAL 1107HYGAS. Plants were regenerated fromsaid cells.

EXAMPLE 6

In this example, we transform a plant of the species Brassica napus witha recombinant DNA molecule comprising a ssquence coding for the Ricin Achain toxin and a pollen specific promoter derived from Brassica napus(clone L 4). A published sequence of a Ricinus communis agglutinin gene(Roberts et al., JBC, 260:15682-15686) was used to construct a ricinspecific probe and isolate the ricin gene from a genomic library ofRicinus zanzibarensis DNA. A DNA fragment coding for the mature A chainsequence (amino acid 2 through 262) was generated by exonucleasedigestion of a 2.3 kb Eco RI restriction endonuclease fragment thatcontained the entire A chain coding sequence and a portion of the Bchain of ricin subcloned as an Eco RI restriction endonuclease fragmentin pGEM 4Z. The construction of the clone containing the A chain codingregion is shown in FIG. 4. Deletion of the coding sequences of theB-chain ricin was done as follows. The subclone was cut with Kpn Irestriction endonuclease and Sac I restriction endonuclease andunidirectionally digested with Exonuclease III. The digested plasmid wastreated with S1 nuclease and Klenow fragment, religated in the presenceof a universal translation terminator (purchased from Pharmacia P-LBiochemicais, Dorval, Quebec, Canada) which has translation stop codonsin all three reading frames. Deleted subclones were recovered andindividual subclones were sequenced. One such subclone was found tocontain a deletion that encompassed the internal portion of the B chainupstream to the codon coding for amino acid 262 (proline) of the A chainin the published sequence. The sequence of the deletion end point,reading 5' to 3', is as follows: ##STR1##

As shown above, the polylinker contains a Bam HI restrictionendonuclease site. We also determined that there was a Bam HIrestriction site upstream from the mature A chain in this clone. Thisdeletion subclone was digested with Bam HI restriction endonuclease.This Bam HI restriction endonuclease fragment which codes for the Achain of Ricin was subcloned into Bam HI restriction endonuclease cutpGEM-3. The inserted fragment was positioned so that the 5' end of thericin A chain gene was next to the Xba I restriction endonuclease sitein pGEM 3. This subclone was unidirectionally deleted with ExonucleaseIII following digestion of the clone with Pst I and Sal I restrictionendonucleases. Following digestion, the digested DNA was treated with S1nuclease and Klenow fragment, and was religated in the presence of apalindromic oligonucleotide linker (sequence being:5'-CATCGGATCCGATG-3') such that the entire deletion could be excisedfrom the plasmid with Bam HI restriction endonuclease and contains anATG initiation codon. Deletion subclones were picked and sequenced andone was chosen that was deleted to amino acid residue 2 and had thesequence (reading from polylinker into the 5' end of the gene) 5-CCC GGGGAT CCG ATG TTC-3' whereas the ATG codon specifies an initiation codonin frame to the mature A chain sequence and was introduced by theinsertion of the palindromic oligonucleotide linker and the last threenucleotides of that sequence code for a phenylalanine amino acid residuethat is the second residue in the mature A chain of ricin. This wasdeduced by comparison of the sequence with previously published reportsof the sequence of ricin. This clone was named pPALAC and contained thericin A chain gene excisable as a Bam HI fragment. This Bam HIrestriction endonuclease fragment was subsequently cloned into PAL 1107by the use of the single Bam HI restriction endonuclease site of thepolylinker of PAL 1107. The 3' end of this A chain gene contained at its3' end the three frame translation terminator such that only the A chainprotein would be produced by this construct wherein the C terminal aminoacids are: pro-ala-(stop). In between these newly created N-terminal andC-terminal amino acid residues was the mature ricin A chain amino acidsequence as coded for by the deleted ricin gene. Vectors with thisrecombinant ricin gene in the opposite (antisense) orientation were alsorecovered (such vectors containing the antisense recombinant ricin geneare referred to as PAL 1107RICAS). Clones containing the recombinantricin gene under control of the microspore specific promoter in PAL 1107and in the sense orientation were named PAL 1107RIC.

The PAL 1107RICAS and PAL 1107RIC were used to transform plants of thespecies Brassica napus according to the method described in example 1.

EXAMPLE 7

In this example, we use the same procedure used in example 6, however,we use a truncated version of Ricin A chain gene.

A truncated version of the ricin A chain gene of pPAL-AC was isolated bydigesting pPAL-AC with Bam HI and Bgl II. This releases an A chainfragment containing a Bam HII site preceeding the ATG start, and thefirst 196 amino acid codons of the mature A chain sequence. Thisfragment was cloned into the Bam HI site of PAL 1107 and a clonecontaining the gene in the sense orientation was recovered. This clone(PAL 1107 containing the truncated A chain gene) was digested with SmaI, phosphorylated with alkaline phosphatase and religated in thepresence of the universal translation terminator described in example 6.Clones containing this terminator inserted into the Sma I site wererecovered and were named PAL 1107 AC. PAL 1107AC was used to transformBrassica napus stem epidermal layer peels as described in example 1.

EXAMPLE 8

In this example, we use the same procedure used in example 7 totransform tobacco.

The vector PAL 1107AC was used to transform tobacco leaf discs asdescribed in example 3. Transformed plants containing the truncatedricin A chain gene were recovered.

EXAMPLE 9

In this example, we use the same procedure used in example 7, totransform tomato.

The vector PAL 1107AC was used to transform tomato leaf discs asdescribed in example 5.

EXAMPLE 10

In addition to the procedure used in tobacco in example 8, in thisexample, we construct an antisense coding sequence of the Ricin A chaingene and regulate its expression using the same pollen specificpromoter.

The vectors PAL 1107RIC and PAL 1107RICAS were used to produce malesterile and restoration lines in tobacco.

Tobacco leaf discs were transformed with these two vectors usingprocedures outlined in example 3. Transformed regenerated plantscontaining the antisense and sense ricin A chain genes were recovered.

EXAMPLE 11

In this example, we repeat the procedure used in example 10 with tomato.

The vectors PAL 1107RIC and PAL 1107RICAS were used to produce malesterile and restorer lines in tomato. Tomato leaf discs were transformedwith these two vectors using procedures outlined in example 5.

EXAMPLE 12

In this example, we inactivate a pollen specific gene derived fromBassica napus (clone L 19) using antisense DNA under the control of theCaMV 35S constitutive promoter.

The use of antisense RNA to control fertility can also be accomplishedby the use of a promoter that is functional in all tissues such that anygene placed under control of this promoter will be transcribed in allcells of the plant. In this example, the Cauliflower Mosaic Virus 35Spromoter in the plasmid pRAJ 221 was used to produce antisense RNA bydirecting the transcription of the 1.3 Kb Hind III restrictionendonuclease fragment containing coding sequence from L 19 cloned in theantisense orientation to this promoter. This was accomplished bydigesting pRAJ-221 with Hind III and Xba I and inserting this CaliflowerMosaic Virus 355 promoter fragment into Hind III-Xba I cut PAL 1001described above. This produced a vector (PAL 1007) into which fragmentscan be conveniently cloned in the portion of the poiylinker from BIN 19located between the Cauliflower Mosaic Virus 35S promoter and the noster. Sma 1 was used to cut PAL 1007 and the 1.3 Kb Hind III fragmentfrom clone L 19 was isolated, made blunt end by Klenow treatment andinserted into this Sma I site of PAL 1007. Clones containing thefragment in the antisense orientation were chosen and used to transformBrassica napus thin stem epidermal layers of cv. Westar as outlined inexample 1.

EXAMPLE 13

In this example, we inactivate a different pollen specific gene derivedfrom Brassica napus (clone L 4) using antisense DNA under the control ofCaMV 35S constitutive promoter.

The use of antisense RNA to control fertility can be accomplished usinga promoter functional in all tissues such as that described in example12. The vector PAL 1107 was cut with Bam HI and Sma 1. To this vectorwas added the 1.68 Kb Bam HI-Hinc II fragment of clone L 4. This vector(PAL 1311) carries the coding region of L 4 in the antisenseorientation. PAL 1131 wa used to transform Brassica napus stem epidermallayer peels as described in example 1.

EXAMPLE 14

In this example, we inactivate a pollen specific gene derived fromBrassica napus (clone L 19) using antisense DNA under the control of theinducible heat shock promoter fragment of the Drosophila HSP 70 gene.

The inducible heat shock promoter fragment of the Drosophila HSP 70 genein pPW 229 (obtained from. Meselson, M., Harvard University anddescribed in Livak et al., 1978 Proc. Natl. Acad. Sci. USA 75:5613-5617)was isolated as a Hind III-Pst I restriction endonuclease fragment andcloned into Hind III-Pst I restriction endonuclease cut pGEM 4Z. Theheat shock promoter fragment was excised as a Hind III-Sma I fragmentand cloned into Hind III-Sma I cut PAL 1001. This produced a vector (PAL1009) that contains a heat shock promoter followed by a portion of thepolylinker and the nos ter. The Sma I restriction endonuclease site inPAL 1009 was used for the cloning of a 1.3 Kb Hind III restrictionendonuclease fragment containing coding sequence from L 19 following themaking of this 1.3 Kb fragment blunt end with Klenow fragment. The clonecontaining sequence in the antisense orientation was determined byrestriction mapping. This vector was named PAL 1403. This vector wasused for transformation of Brassica napus, following procedures detailedin example 1.

EXAMPLE 15

In this example, we inactivate the actin gene, a gene that is criticalto cellular function and development in all metabolically competentcells. We use an antisense version of said actin gene under the controlof a pollen specific promoter derived from Brassica napus (clone L 4).

The use of a pollen specific promoter to direct the synthesis ofantisense RNA causing male sterility was accomplished by inserting anactin gene in the antisense orientation under the control of the pollenspecific promoter in PAL 1107 by using pSAc3, a soybean actin clone toprovide an actin coding region. (pSAc3 was provided by R. Meagher) Thecoding region of actin was isolated as a Eco RI-Tag I fragment madeblunt ended and inserted into Sma I cut PAL 1107. Clones containing thegene in the antisense orientation were chosen and named PAL 1107ASac.This vector was used to transform Brassica napus stem epidermal layerpeels using the method outlined in example 1.

EXAMPLE 16

In this example we use the same procedure used in example 15 withtomato.

PAL 1107ASac was used to transform tomato leaf discs using the method ofexample 5.

EXAMPLE 17

In this example, we use the same procedure used in example 15 withtobacco.

PAL 1107Sac was used to transform tobacco leaf discs using the methoddescribed in example 3.

We claim:
 1. A method of producing hybrid seed from plants of a speciesof pollen-producing plants that is capable of being geneticallytransformed, comprising the steps of:(a) producing a geneticallytransformed plant by:(i) inserting into the genome of a plant cell ofsaid species a recombinant DNA molecule comprising(I) an antisense DNAsequence the transcript of which renders developing pollen grainssusceptible to an herbicide or antibiotic and (II) a promoter thatfunctions in said plant cell to cause transcription of said antisenseDNA sequence into RNA preferentially in cells critical to pollenformation or function, (ii) obtaining a transformed plant cell of saidplant and (iii) regenerating from said transformed plant cell a plantthat is genetically transformed with said recombinant DNA molecule suchthat said plant produces said RNA to render developing pollen grainssusceptible to said herbicide or antibiotic; (b) increasing the numberof genetically transformed plants by:(i) fertilizing said geneticallytransformed plant with pollen produced by a suitable male fertile plantand obtaining seed which, when germinated, yields a plurality ofgenetically transformed plants, or (ii) clonally propagating saidgenetically transformed plant to obtain a plurality of geneticallytransformed plants; and (c) effecting a hybrid cross by pollinating saidgenetically transformed plants with pollen from suitable male fertileplant donors.
 2. The method of claim 1, wherein said herbicide isglyphosphate or chlorsulfuron.
 3. The method of claim 1, wherein saidantibiotic is selected from the group consisting of kanamycin, G418 andhygromycin.
 4. The method of claim 1, wherein said antisense DNAsequence is transcribed in developing microspores.
 5. The method ofclaim 4, wherein said developing microspores are early uninucleatemicrospores.
 6. The method of claim 1, wherein said pollen-producingplant is of a genus in the family Cruciferae.
 7. The method of claim 6,wherein said genus is Brassica.
 8. The method of claim 1, wherein saidspecies is Brassica napus or Brassica campestris.
 9. The method of claim1, wherein said pollen-producing plant is of the family Solanaceae. 10.A transformed plant containing a recombinant DNA molecule whichcomprises (1) an antisense DNA sequence the transcript of which rendersdeveloping pollen grains susceptible to an herbicide or antibiotic, and(2) a promoter that functions in said plant cell to cause transcriptionof said antisense DNA sequence into RNA preferentially in cells criticalto pollen formation or function.
 11. Hybrid seed containing arecombinant DNA molecule which comprises (1) an antisense DNA sequencethe transcript of which renders developing pollen grains susceptible toan herbicide or antibiotic, and (2) a promoter that functions in saidplant cell to cause transcription of said antisense DNA sequence intoRNA preferentially in cells critical to pollen formation or function.12. The method according to claim 1, wherein said promoter causestranscription preferentially in pollen.
 13. The method according toclaim 1, wherein said promoter causes transcription preferentially indeveloping microspores.
 14. The method according to claim 1, whereinsaid promoter causes transcription preferentially in anther cells. 15.The plant according to claim 10, wherein said promoter causestranscription preferentially in pollen.
 16. The plant according to claim10, wherein said promoter causes transcription preferentially indeveloping microspores.
 17. The plant according to claim 10, whereinsaid promoter causes transcription preferentially in anther cells. 18.The seed according to claim 11, wherein said promoter causestranscription preferentially in pollen.
 19. The seed according to claim11, wherein said promoter causes transcription preferentially indeveloping microspores.
 20. The seed according to claim 11, wherein saidpromoter causes transcription preferentially in anther cells.